Laboratory Techniques in Biochemistry and Molecular Biology Vol 08: Cell Culture for Biochemists

Cell culture for biochemists

LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY Volume 8 Edited by R.H. BURDON - Department of Biochemistry, University of Strathclyde, Glasgow

P.H. van KNIPPENBERG - Department of Biochemistry, University of Leiden, Leiden

ELSEVIER AMSTERDAM. NEW YORK . OXFORD

CELL CULTURE FOR BIOCHEMISTS

Second revised edition

R.L.P. Adams Department of Biochemistry University of Gfasgow Gfasgow GI2 SQQ Scotland, U.K.

1990 ELSEVIER AMSTERDAM. NEW YORK . OXFORD

0 1990,

Elsevier Science Publishers B. V. (Biomedical Division)

AI rights reserved. No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science Publishers B. V. (Biomedical Division), P.O. Box 1527, lo00 BM Amsterdam, The Netherlands. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of the rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made. Special regulations for readers in the U.S.A.: This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which the photocopying of parts of this publication may be made in the U.S.A. All other copyright questions, including photocopying outside the U.S.A., should be referred to the publisher. ISBN 0-444-81297-0 (paperback) ISBN 0-444-81306-3 (hardback) ISBN 0-7204-4200-1 (series) 1st edition 2nd printing 3rd printing 4th printing

2nd revised edition 1990

1981 1983 1985 1988

Published by: ELSEVIER SCIENCE PUBLISHERS B.V. (BIOMEDICAL DIVISION) P.O. BOX 211 lo00 AE AMSTERDAM THE NETHERLANDS Sole distributorsfor the U.S.A. and Canada: ELSEVIER SCIENCE PUBLISHING COMPANY, INC. 655 AVENUE OF THE AMERICAS NEW YORK, N Y 10010 U.S.A. Library of Congress Card No. 85-647011

Printed in The Netherlands

List of abbreviations 2,4-D ACTH Ad2 AMP, ADP, ATP ATCC BHK cells BME BPL BRL BSA BSS BUdr CHO cells CMF CMP, CDP, CTP

2,4-diphenoxyacetic acid adrenocorticotrophic hormone adeno 2 virus adenosine monophosphate, diphosphate, triphosphate American Type Culture Collection baby hamster kidney cells basal medium (Eagle) P-propiolactone Buffalo rat liver bovine serum albumin balanced or buffered salt solution bromodeoxyuridine Chinese hamster ovary cells calcium- and magnesium-free BSS

cytidine monophosphate, diphosphate, triphosphate Connaught Medical Research Laboratories CMRL Calgon metasilicate CMS cytopathic effect CPE dAMP. dTMP deoxyadenosine monophosphate, deoxythymidine monophosphate, etc. 2-diamidino-phenyl indole DAPI a detergent Decon mounting medium available from G.T. Gurr Ltd. DePex (Appendix 3) Eagle’s MEM supplemented with 10% calf serum EClO ethylenediaminetetraacetic acid (versene) EDTA

v1

EFC EGF EMS ESG ETC FBS FGF FITC GO-phase G1-phase GZphase GMP, GDP, GTP GS-filter HAT medium HAU HECS Hep cells HPRT HSV HTC cells IAA ITES M MCDB

MEM Methocel MNNG m.0.i. MOPC

CELL CULTURE FOR BIOCHEMISTS

Eagle’s MEM supplemented with foetal calf serum epidermal growth factor ethyl methane sulphonate Ewing sarcoma growth factor Eagle’s MEM supplemented with tryptose phosphate (10%) and calf serum (10%) foetal bovine serum fibroblast growth factor fluoroscein isothiocyanate the resting stage of the cell cycle the first gap in the cell cycle (between M and S) the second gap in the cell cycle (between S and M) guanosine monophosphate, diphosphate, triphosphate 0.22 pm membrane filter supplied by Millipore COrP medium supplemented with hypoxanthine, aminopterin and thymidine haemagglutinin unit human endothelial cell supernatant human epithelial cells hypoxanthine phosphoribosyl transferase herpes simplex virus hepatoma tissue culture cells indole acetic acid medium supplement containing insulin, transferrin, ethanolamine and selenium mitosis media developed at the Department of Molecular, Cellular and Developmental Biology, University of Colorado minimum essential medium carboxymethyl cellulose type MC (4000 centripoises) sold by Dow Chemical Co. N-methyl-N-nitro-N-nitrosoguanidine multiplicity of infection mineral oil-induced plasmacytoma

LIST OF ABBREVIATIONS

MSE MVM NCTC NDV PBS PCA p.f.u. PGE PHA PPLO PPO p.s.i. PyY cells RPMI RSV SDS S-phase

ssc

SV40 T3 T-antigen TC TCA tG1, rG2, I S and rM TK TRITC ts UMP, UDP, UTP

vsv 7x

Vii

Medical and Scientific Equipment Ltd. minute virus of mouse National Cancer Tissue Culture Newcastle disease virus phosphate buffered saline perchloric acid plaque forming unit prostaglandin E ph ytohaemagglutinin pleuropneumonia-like organism diphenyloxazole pounds per square inch (14.7 p.s.i. = 1 atmosphere = lo5 Pascal) polyoma transformed BHK cells Roswell Park Memorial Institute Rous sarcoma virus sodium dodecyl sulphate DNA synthetic phase of cell cycle saline sodium citrate simian virus 40 t riiodot hyronine transplantation antigen tissue culture trichloracetic acid the time for the cell phases G1, G2, S and M, respectively thymidine kinase tetramethylrhodamine isothiocyanate temperature sensitive uridine monophosphate, diphosphate, triphosphate vesicular stomatitis virus a detergent

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Acknowledgements I would like to sincerely thank the following from the University of Glasgow: the staff of the Wellcome Cell Culture Unit for the help given for many years; the staff of the Medical Illustration Unit for most of the illustrations in this book; the Secretaries of the Biochemistry Department for typing and retyping my notes with only the occasional cry of anguish and Mrs. Angela Rinaldi for invaluable technical assistance. Thanks are also due to all those who have given permission for their figures to be used and to Professor Houslay and the late Professor Smellie for support, and to the Medical Research Council and the Cancer Research Campaign whose financial support of my research has enabled me to continue to use the Cell Culture Unit which was endowed initially through a grant from the Wellcome Foundation.

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Contents List of abbreviations . . . . . . . . . . . . . . . . . . . . . .

.................

.........................

0

.

ix

...........

1

1.1. Background . . . . . . . . . . . . . . . . . . . . . ................. ........ 1.2. Some advantages . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................. 1.3.1. Differentiation . . . . . . . . . . . 1.3.2. Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... 1.3.3. Immunology . . 1.3.4. Hormones and growth factors . . . . . . . . . 1.3.5. Virology and cell transformation . . . . . . . . . . . . . . . . . . . . . . ................. 1.3.6. Cytotoxicity testing . . . . . . . . . . . 1.4. Animal cell biotechnology . . . . . . . . . . . . . . . . . . . .

1 2 4 4

A cknowledgemenls

Chapter 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . .

5 6 7 8

.......

I!

2.1. Types of cells . . . . . . . ........................ 2.2. Primary cells and transf ... ................. 2.3. Growth control . . . . . . . . . . . . . . . . . . . . . . . . . . ........ 2.3.1. Nutritional requirements . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Cell cycle and growth cycle . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Attachment and spreading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Anchorage dependence and growth in suspension . . . 2.4.2. Density dependent reg .................... .......... 2.4.3. 2.5. Growth .... ........ 2.5.1. onent ........ ............. 2.5.2. Growth factors for hae 2.5.3. Mechanism of action o s ............ 2.5.4. Oncogenes . . . . . . . . . . . . . . . . 2.5.5. Steroid hormones . . . . . . . . . . . . . . . . . 2.6. Differentiated functions in cell cultures . . . . . . . .

11 13 16 16 18 19

Chapter 2. Characteristics of cultured cells . . . . . . . . . . . .

21 26 21

..................................

35

3.1. Design of culture vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Gaseous exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35 35

Chapter 3. Culture vessels

xii

3.2.

C E L L C U L T U R E FOR BIOCHEMISTS

3.1.2. Sealed vessels ........................... ............................... 3.1.3. Perfusion tech Small scale cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

............................. ................................. ................................. 3.4.2. Cell factories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. Perfusion vessels ............................ 3.4.4. Capillary beds . . ............................ 3.5. Suspension cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Microcarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s

3.6.1. Positively charged microcarriers . . . . . . . . . . . . . . . . . . . . . . . ....... 3.6.2. Negatively charged microcarriers 3.6.3. Collagen or gelatin (denatured collagen) beads . . . . . . . . . . . . 3.6.4. Cell entrapment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Air lift systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 4. Subculturing

.......................................

Dissociation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Trypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Pronase . . . . . . . . . . . . . . . . 4.1.3. Collagenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. Dispase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.5. EDTA (Versene) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.6. Mechanical means 4.2. Subculture of a cell monolayer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Viable cell count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Bacterial check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Subculture of cells growing in suspension 4.4. Protocol for setting up microcarrier cultures . . . . . . . . . . . . . . . . . . . 4.4.1. Preparation of the microcarrier ........... 4.4.2. Preparation of the culture vessel . . . . . . . . . . . . . . . . . . . . . . 4.4.3. Initiating a culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.4. Non-stirred microcarriers 4.5. Subculture of cells growing on microcarriers . . . . . . . . . . . . . . . . . . . 4.6. The growth cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.

Chapter 5. Cell culture media

....................................

5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Balanced salt solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Zwitterionic buffers 5.3. Eagle’s medium . . . . . . . . . 5.3.1. Powdered media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. More complicated media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Simple media with unspecified additives 5.6. Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

36 38 38 41 41 41

44 45 46 51 52 54 55 55 56 59

59 59 60 60 61 63 64 64 64 65 65 65 66 66 67 68 71

71 72 75 75 77 78 79 79 80

...

CONTENTS

XlU

5.7.1. Removal of small molecules from serum . . . . . . . . . . . . 5.8. Serum-free media ..............................

84

................... 5.8.2. Defined media . . . . . . . . . . . . . . . . . . . . . . . . 5.8.3. Media for isolation of secreted products 5.8.4. Commercial media . . . . . . . . . . . . . . . 5.8.5. Isolation of factors from culture superna 5.9. Media for culture of insect cells . . . . . . . . . . . . . . . 5.10. Media for culture of plant cells . . . . . . . . . . . .

......

Chapter 6. Primmy cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.1. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Lymphocytes ................. 6.2.1. Isolation of leukocytes and autologous plasma . . . . 6.2.2. Purification of lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.3. Glass-bead column method . . . . . . . . . . . . . 6.2.4. Gradient centrifugation method . . . . . . . . . . . . . . . . . . . . . . . 6.2.5. Cultured lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Human skin biopsies . . . . . . . . . . . . . . . . . 6.4. Mouse or rat embryo cultures . . . . . . . . . . . 6.5. Chick embryo cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Chick embryo liver cells . ............... 6.7. Rat hepatocytes . . . . . 6.8. Primary kidney cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.9. Endothelial cells . . . . . . . . . . . . . . . . . . . . . 6.10. Mammary epithelial cell cultures . . . . . . . . . ............... 6.11. Colonic epithelial cells . 6.12. Rat or chick skeletal mu ........ 6.13. Mouse macrophage cultures . . . . . . . . . . . . . . . . . . . . . . . . 6.14. Ascites cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.15. Dipteran cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 7. Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.1. Cell cloning and plating efficiency . . . . . 7.1.1. Measurement of plating efficiency . . . . . . . . . . . 7.1.3. Cloning under agar

.........

......................

........ 7.3.1. Freezing procedure 7.3.2. Storage procedure

. . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . .. .. .. .. .. .. ........... .. .. .. .. .. . . . . . . . . . .

7.3.4. Organisation of stocks of frozen cells . . . . . . . . . . . . . . . . . . . Cell banks and transport of cells cells . . . . .. ......... . . . . . . . . . . . .. . . 7.3.5. Cell

93

97

97 97 98 98 98 99 100 105 106 108 112

114

I I7 117 120

123 129

135 135 135

xiv

CELL CULTURE FOR BIOCHEMISTS

7.4. Karyotyping . . . . . . . . . . . . . . . . . 7.4.1. Chromosome preparation . . . . . . . . . . . . . . . . . . . . . . . 7.4.2. Karyotyping ................................

138

.........................

140

7.5.1. Calcium phosphate method . . . . . .

...............

7.6. Cell visualisation . . . . . . . . . . . . . . 7.6.1. Phase contrast microscopy . . . . . . . . . . . . . . . . . . . . . . . . . .

142

147

7.7. Sub-cellular fractionation . . . . . . . . . . . . . . . . . . Chapter 8.Glassware preparation and sterilisation techniques

......

8.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.1. Waslung procedure ............... ............... 8.1.2. Bottles and pipettes 8.1.3. Rubber stoppers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Sterilisation by heat.

....................................

8.2.3. Control of sterilisation . . . . 8.3. Sterilisation by filtration . . . . . . . . 8.3.1. Self-assembled filter apparatus . . . . . . . . . . . . . . . . . . . . . . . 8.3.2. Disposable filter apparatus . . . . . . . . . . . . . . . . . . . . . . Chapter 9. Contamination

......................................

9.1. Bacterial contamination . 9.1.1. Glass and plasticw 9.1.2. Cells . . . . . . . . . 9.1.3. M e d i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Sterilitychecks . . . . ..... .... 9.3. Analysis of bacterial contamination . . . 9.4. Airborne contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.1. Aseptic technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2. Laminar flow syst 9.5. Antibiotics . . . . . . . . . . 9.6. Disposal of contaminated material . . . . . . . . . . . . . . . . . . . . . . . . . . .... 9.7. ......... 9.7.1. Effect on cell cultures . . . . . . . . . . . . . . . 9.7.2. Culture of mycoplasmas . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ .... 9.8. Testing for mycoplasma . 9.8.1. Orceinstain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.8.2. Autoradiography . . . . . . . . . . . . . . . . . . . . ..... .. . . . . . .. .. .... . . 9.8.3. Fluorescence staining . . .. ..... . . . . . . . . . .. . . . . . . . . . . . . .. .. . .

151 152 152 153 153

156

165

166 168 168 169 174 174 176 176 177 178 178 179

xv

CONTENTS

9.8.4. Cell growth test for mycoplasmas . . . . . . . . . . . . . . . . . . . . . . 9.8.5. DNA probe for mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . 9.9. Elimination of mycoplasmas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.10. Viral contamination . . . . . . . . . . . , . . . . . . . . . . . . , . . . . . . . . . . .

182 183 183 184

. . . . . . ..

187

10.1. 10.2. 10.3. 10.4.

Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitosis . . . . . . _ . . .. . . ... . . . . . . ... . .. . . . . . S-phase . . . . . . Control of the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4.1. The GO-phase and commitment to cycle .... 10.4.2. P34 and cyclins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5. Distribution of cells around the cycle . . . . . . . . . . . . . . . . . . . . . . . . 10.6. Growth fraction . . . . . . . . . . . . . .. .. 10.7. Cell cycle analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7.1. Tritiated thymidine pulse method . . . . . . . . . . . . . . . . . . . . . . 10.7.2. Continuous labelling method . . . . . . . . . 10.7.3. Accumulation functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...,... .. 10.7.4. Graphical analysis . . . 10.7.5. Flow microfluorometry (FACS) . . . . . . . . . . . . . . . . . . . . . . .

187 188 190 191 191 195 196 199 199 200 201 203 206 207

Chapter 11. Cell synchronisation . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . .

21 I

...... Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... Selection of mitotic cells . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . 11.2.1. Shaking . . . . 11.2.2. Trypsinisation . 11.2.3. Selection from microcarriers . . . . . . . . . . . . . . Selective killing of cells in particular phases . . . . . . . . . . . . . . . . . . . . . Selection of cells by sue . . . . . . . . . . 11.4.1. Electronic cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.2. Zone sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ . 11.4.3. Centrifugal elutriation . . . . . . . . . . . . . . ...... . . Synchronisation by su Serum deprivation . . .................. Isoleucine starvation .._............... Blocka of S-phase . . . . . . . . . . . . . . . . .. ...... . . . . . . . . . . Blockade . 11.8.1. Action of aminopterin and amethoptenn thoptenn (methotrexate) (methotrexa ..... 11.8.1. 11.8.2. Action of 5-fluorodeoxyuridine . . . . . . . . . . . . . . . . . . . . . . . .. 11.8.3. 11.8.3. Action of high concentrations of thymidine . . 11.8.4. 11.8.4. Action of hydroxyurea . . . . . . . . . . . . . . . .... ........... .. . . . .. . . . Procedure for inducing synchrony at the Gl/S interphase . . . . . .. . .. . . rphase.. Proced 11.9.1. Isoleucine starvation and hydroxyurea . . . 11.9.1. .. . 11.9.2. Stationary phase cells and a 11.9.2. ........... 11.9.3. ................ 11.9.3. Double thymidine block . . 11.9.4. 11.9.4. Comparison of the methods . . . . . . . . . . . .. ....... .. . . . .. ....... .. . . . Synchr Synchronisation in G2 . . . . . . . . . . . . .. .. .. .. ........................ . . . . . . . Synchronisation in M . . .. ....... .. . . . . .. ....... . . . . .......... . . . . . . . . . . ..

211 212 212 213 214 215 215 215 216 219 222 224 226 328 229 232 232 235 235 235 236 236 236 237 238

Chapter 10. The cell cycle . . . . . . . . . .

11.1 11.2

11.3. 11.4.

11.5. 11.6. 11.7. 11.8. 11.8.

11.9. 11.9.

11.10, 11.10. 11.11, 11.11.

Xvi

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Chapter 12. Use of radioactioe isotopes in cell culture . . .

239

12.1. Estimation of rates of DNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . 12.1.1. Flooding the pool 12.1.2. Blocking the endo 12.1.3. Allowing for endogenous dTTP . . . . . . . . . . . . . . . . . . . . . . . 12.1.4. Comparison of the methods . . . . . . . . . . . . . . . . . . . . . . . 12.1.5. Application to suspension cultures . . . . . . . . . . . . . . . . . . . . . 12.2. Estimation of rates of RNA and protein synthesis . . . . . . . . . . . . . . . 12.3. Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.1. Fixation and staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.2. Emulsions . . . . . . . . . . . . . 12.3.3. Stripping film . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.4. Liquid emulsion ....................... 12.3.5. Autoradiography in dishes . . . . . . . . . . . . . . . . . . . . 12.3.6. The value of grain counting . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.7. Background grains . . . . . . . . . . . . . . 12.3.8. Autoradiography of water-soluble cell components . . . . . . . . . 12.3.8.1. Cell fixation 12.3.8.2. Covering with emulsion . . . . . . . . . . . . . . . . . . . . . . 12.4. Labelling with bromodeoxyuridine . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5. DNA repair . . . . . 12.4.2. Estimation of repair synthesis . . . . . . . . . . . . . . . . . . . . . . . .

239 243 245 246 247 248 249 250 251 251 252 253 255 255 256 257 257 258 259 259 260 261

...........................

263

Chaprer 13. Cell mutants and cell hybrids

13.7. Methods of fusion ................... 13.7. ..... 13.7.1. Cell fusion eglycol . . . . . . . . . . . . . . . . . . . . 13.7.2. Cell fusion 13.7.3. Electrofusi ......................... 13.8. Cell communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.8.1. Grain counting and cell communication . . . . . . . . . . . . . . . . .

263 264 266 267 267 268 268 269 271 272 273 273 273 273 274 275 276

Chapter 14. V i m e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279

14.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1.1. Animal virus classification . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1.2. Precautions to be taken when using virus infected cells . . . . . . .

279 280 280

13.1. Auxotrophic mutants . 13.2. Selection of mutants . 13.2.1. Procedure for isolation of TK- mutants . . . . . . . . . . . . . . . . . 13.3. 13.3.1. Selection of G1 mutants and S mutants . . . . . . . . . . 13.3.2. Selection of G2 mutants and M mutants . . . . . . . . . . . . . . . . . ells . . . . . . . . . . . . . . . . 13.4. ........ 13.4. Replica plating of animal cells 13.5. Somatic cell hybridisation . . . ....... . . . .......... . . . ....... . 13.5. a1 antibody production 13.6. 13.6.1. Isolation of spleen cells . . . . . . . . . . . . . . . . . ........

CONTENTS

xvii

14.2. Virus production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.1. Procedure for production of herpes simplex. pseudorabies or EMCvirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.2. Procedure for production of SV40 virus . . . . . . . . . . . . . . . . . ......... 14.2.3. One-step growth curve of SV40 . . 14.2.4. Sendai virus - production and inactivation . . . . . . . . . . . . . . . 14.2.4.1. Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.4.2. Inactivation by UV . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.4.3. Inactivation by P-propiolactone . . . . . . . . . . . . . . . . 14.3. Virus detection 14.3.1. Plaque assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.1.1. Viral dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.1.2. Suspension assay . . . . . . . . . . . . . . . 14.3.1.3. Monolayer assay . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.1.4. Agar overlay assay . . . . . . . 14.3.2. Fluorescent antibody techniques . . . . . . . . . . . . . . . . . . . . . . 14.3.2.1. Preparation of antisera . . . . . . . . . . . . . . . . . . . . . . 14.3.2.2. Preparation of globulin fraction 14.3.2.3. Conjugation of antisera with fluo 14.3.2.4. Staining techniques . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.3. Haemadsorption and haemagglutination 14.4. Production and testing of viral vaccines . . . . . . . . . . . . . . . . . . . . . . . 14.5. Viral transformation of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5.1. Methods of transformation ................

283 284 286 287 287 287 287 288 288 289 289 289 290 292 293 293 293 294 295 296 297 298

Chapter I5. Di/freniiation in cell cultures . . . . . . . . . . . . . . . . . . . . . . . . . . .

301

15.1. Erythroid differentiation of Friend cells . . . . . . . . . . . . . . . . . . . . . . 15.1.1. Induction of globin synthesis in Friend cells ......... 15.2. Skin and keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3. Teratocarcinoma cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4. Differentiation of muscle cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5. Differentiation of adipose c 15.6. Differentiated hepatoctyes

301 302 303 305 307 308 308

Chapter I6. Appendices

1. 2. 3. 4. 5.

...........

Media formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stains and fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppliers ..... Sterility checks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

283

311

311 325 327 331 333

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

335

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355

Subjectindex

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CHAPTER 1

Introduction 1.I. Background Although a number of books are available giving a great deal of information about various aspects of cell culture this book is designed rather for the biochemist or molecular biologist, whose interest in cell culture extends only as far as this technique provides him with material with which he may perform biochemical experiments. This is the second edition of this book and, although some sections have been extensively revised, others are little changed. For instance the details of glassware preparation have not changed over the last 10 years but what has changed is the use of glassware which has been superseded almost entirely with presterilised plastic ware. The aspects which have changed most over the last 10 years are those which result from a greater understanding of the controls over cell division, e.g. the role of growth factors and the use of serum-free medium. Before a biochemist will apply himself to using the technique of cell culture he must be assured that it offers him significant advantages which outweigh any disadvantages. Furthermore, he must not imagine that the methods are too laborious for routine use or that some degree of black magic is required before success can be achieved. To some extent such fears are based on the experience of workers in the field up to about 1960. In the preceding 30 years nearly all major cell types had been cultivated for varying periods and much descriptive information obtained but this was only as a result of constant dedicated effort. Since 1960 many of the obstacles have been removed from the path of the biochemist. Perhaps the most important is that commercial companies now supply media, sera, cells and culture vessels 1

2

CELL CULTURE FOR BIOCHEMISTS

which enable cells to be cultured occasionally or routinely on a scale varying from a growth surface of less than one square centimetre up to several square metres. This service is available only as a result of the description of simple media in which the cells grow well and the development of simple methods for isolation of primary cells, selection of clones and storage of cell lines. The other major fear is one of cost. On a weight for weight basis cultured cells are several orders of magnitude more expensive than, say, rat liver. Thus a rat costs under f 5 and yields about 10 g of liver. lo6 cells obtained from a commercial supplier cost about f 2 and so 10 g of cells (2 X 10" cells) could cost f40,OOO although significant price reductions are obtained when buying in quantity. One can produce the same number of cells in one's own laboratory for less (the cost of medium may be as little as E25), but this hides the cost of overheads. Nevertheless, the use of cultured cells must offer marked advantages before it is worthwhile to embark on large scale production, and there is seldom any justification for using cultured cells as a source of material for an enzyme purification when sources like rat liver or rabbit kidney would do as well. At the other extreme, however, many experiments may be performed with 102-106 cultured cells at a cost equivalent to the alternatives.

I . 2. Some advantages One major advantage offered by cultured cells which cell biologists make full use of but which is often ignored by biochemists is that the living cells may be watched under the microscope. It is essential that healthy cells are used in an experiment and that they remain alive throughout the experiment. That this is the case may be monitored regularly and moreover quantitative estimates of the proportion of viable cells are readily obtained. It is often impossible to know the state of an animal's kidney until the end of the experiment and then usually only in a qualitative manner. Cells in culture offer a homogeneous population of cells of virtually identical genetic make-up, growing in a constant environment. Moreover, the environment may be changed, withm limits, at

CH. 1.

INTRODUCTION

3

the whim of the experimenter who may thereby investigate the effect of pH, temperature, amino acid and vitamin concentration etc. on the growth of the cells. Growth may be measured over a short time period either by measuring an increase in cell number or size, or by following the incorporation of a radioactive tracer into DNA. These are real advantages over a whole animal system, placing cultured cells on a par with microorganisms as an experimental system. Using cultured cells, the growth requirements of human cells were analysed in a few weeks thus confirming decades of work with people of different genetic background living in different environments (Eagle, 195a,b; see 9 2.3.1). Moreover, significant results may be obtained with very few cells. An experiment which may require 100 rats or 1000 humans in order to clarify some point may be statistically equally valid if 100 coverslip cultures or one microwell plate are used. If each cell is regarded as an independent experiment then one coverslip culture may yield more reliable results than a hospital full of people. This is obviously a major advantage as far as man is concerned but also overcomes the ethical problems which often arise when large numbers of animals are used for experimental purposes. However, in the final analysis, many experiments must be performed on whole animals, but this is no justification for not using cultured cells for the preliminary work. Because cells in culture are easily available for manipulation by the biochemist, radioactive tracers, drugs or hormones etc. may be applied in a known concentration and for a known time period. The amounts of such compounds required may be an order of magnitude less than with comparable experiments on whole animals. There is no fear that the drug whose effect is to be investigated is being metabolised by the liver stored in the muscles and excreted by the kidney. It is usually a simple matter to establish that a substance added to a cell culture remains in contact with the cells in unchanged form at a known concentration for a given time. This enables experiments to yield realistic figures for the rates of incorporation or metabolism of compounds. Such experiments are not without hazards in cultured cells (see Chapter 12) but are very difficult to interpret in whole animals. However, when the aim of the

4

CELL CULTURE FOR BIOCHEMISTS

experiment is to find the effect of a drug or cosmetic on an animal, factors which are problems to one biochemist may be the essence of the experiment to another.

I . 3. Applications Cultured cells have given us great insight into the phenomena of cell growth and differentiation and the general characteristics of the growth of cultured cells are discussed in Chapter 2. It should be clear, however, from reading later chapters that, although the detailed nutritional requirements and growth control mechanisms are complex, it is now a simple matter to culture cells in small or large quantities in order to perform biochemical experiments. Moreover, biochemists are the greatest users of cultured cells and over 60% of cells issued by the Human Genetic Mutant Cell Repository and the Ageing Cell Repository (Camden, N.J.) are issued to biochemists or molecular biologists (Corriell, 1984). 1.3.1. Differentiation

The study of differentiation in higher eukaryotes is extremely difficult, but a number of systems are now available which undergo differentiation in vitro and some of these are considered in Chapter 15. The in vitro systems have the advantage that, following a given stimulus, a population of cells will undergo a change which can be easily recognised and quantitatively monitored. The change may be the production of a protein (e.g. haemoglobin by the Friend cells, see 0 15.1) or more complex alterations in structure and growth pattern such as those occurring during differentiation and fusion of myoblasts (0 15.4) or differentiation of epidermal keratinocytes to form a system resembling the stratum corneum of skin (0 15.2). 1.3.2. Genetics One of the great advantages the bacteriologist had over the traditional biochemist working with eukaryotes was the possession of a

CH. 1 .

INTRODUCTION

5

wide range of mutants which allowed him to perform complex experiments in genetics. The study of familial relationships in eukaryotes was a time consuming occupation and generation times are particularly long for mammals. This problem is accentuated when human genetics is being studied making it at best only an observational science. With the ability to grow cells in culture came techniques enabling cells to be cloned (0 7.1), stored (0 7.3) and fused (0 13.5) which have led to the science of somatic cell genetics. Many of these studies have centred on a gene whose product, hypoxanthine phosphoribosyl transferase (HPRT), is involved in purine nucleotide biosynthesis and a deficiency of which results in gouty arthritis. Cells with a defective HPRT are unable to incorporate the hypoxanthine analogue 8-azaguanine and so are resistant to this analogue’s toxic properties, and cell biologists have exploited the selective advantage of such HPRT- cells in developing techniques of cell hybridisation (0 13.5) and gene transfer (Goss and Harris, 1975; Willicke et al., 1976a,b). Human pedigree studies of families with Lesch-Nyhan syndrome (Seegmiller et al., 1976) showed that the HPRT gene was X-linked, and this was confirmed by analysis of human-mouse cell hybrids which had lost most of their human chromosomes (Ricciuiti and Ruddle, 1973). 1.3.3. Immunology

Although cell culture has been used by immunologists for a number of years the system was beset by a problem. Cells which were synthesising antibodies of interest (e.g. spleen cells from animals injected with specific antibodies) grow poorly or not at all in culture while the myeloma cells produce an antibody of unknown specificity (0 13.6). The ability to fuse these two types of cell has led to the production of monoclonal antibodies on a large scale (Kohler and Milstein, 1975). If a mouse is injected with a crude preparation of an antigen and its spleen cells are subsequently fused to myeloma cells then among the resulting hybrid cells will be one producing a single antibody directed specifically against the antigen. This cell may be cloned (0 7.1) and grown as a tumour in a mouse and in this form

6

CELL CULTURE FOR BIOCHEMISTS

will yield gram quantities of highly specific antibody. Not only is this a help to the immunologist, it also provides the biochemist with antibodies to material he cannot purify, and it has important commercial applications. 1.3.4. Hormones and growth factors From the earlier work discussed at the 1978 Cold Spring Harbor Meeting on Cell Proliferation there has developed a broad field concerned with the study of growth factors and hormones and their effects on cell growth and differentiation. This area is considered in more detail in Chapter 2 and is of enormous clinical importance for the treatment of cancer. As well as the work on peptide hormones much work has focused on the use of MCF-7 and ZR 75.1 cell lines which were isolated from human breast tumour tissue (Lippman et al., 1977; Engel et al., 1978). These cells respond to oestrogen treatment, but the system is not as simple as first thought and may involve paracrine responses (Leake, 1988). 1.3.5. Virology and cell transformation

Much of the rapid progress in the field of virology over the past decades is a consequence of the ability to grow viruses in cells in culture. This not only means that large numbers of animals are no longer required but that assays and procedures which were cumbersome and of poor reproducibility have been replaced with plaque assays, production and staining techniques (Chapter 14) which are simple, accurate and reproducible. This has led to the realisation that viruses not only infect and kill cells but may also bring about the change in cell growth characteristics known as viral transformation of cells (9 2.2 and 9 14.5). These changes, which result in the cell no longer responding to its neighbours in a manner characteristic of the untransformed cell, are being studied in order to throw light on the nature of the transformation event as a similar change in vivo is believed to play a part in the induction of tumours (Q 14.5). As most viral diseases can now be treated by administration of antisera it is important to be able to grow batches of virus both for

CH. 1.

INTRODUCTION

I

identification purposes (0 14.3) and for use in the production of vaccines (0 14.4). Much of this work was done in cultured cells and many hospital virology units are well equipped for growing cells and cultivating viruses on a large scale (see below). 1.3.6. Cytotoxicity testing

A major and increasing application in the use of cell cultures is to test and investigate the mode of action of various products which may be used as drugs, detergents, cosmetics, insecticides and preservatives, etc. Although results obtained using cells in culture cannot be extrapolated directly to the whole animal situation it is fairly certain that if some product produces deleterious effects on several different lines of cells in culture some ill effects may be expected if the product is applied to whole animals. As well as enabling testing to be performed without the possible suffering of large numbers of animals the use of human cells allows testing in one animal species not generally available for experimentation, i.e. man. Moreover, as indicated in 5 1.2, the results of the test are more likely to be reproducible when carried out in vitro. In general, the procedure is to expose cells to a range of concentrations of the drug under test for 24 h and then to test for cell viability (using, for example, neutral red which is taken up only by living cells) or total cellular protein (Goldberg and Frazier, 1989). Such tests are most easily performed in microtitre plates (0 3.2) which allow rapid quantitation of the results using a microtitre plate reader. An alternative method which can also be automated by the use of the Titertek supernatant harvester (see Appendix 3) involves the measurement of radioactive chromium released into the culture medium from killed cells. The harvester consists of a set of absorbent cylinders aligned so that they may be inserted into the wells of a microtitration plate (Appendix 3). Once the supernatant in the wells has been absorbed the cylinders are transferred to counting vials and the amount of radioactive chromium released from the cell monolayer is estimated. Cells take up "Cr sodium chromate rapidly and the excess is readily washed away by rinsing in culture medium.

8

CELL CULTURE FOR BIOCHEMISTS

Labelling need only be for 30 min and on subsequent death of the cell more than 75% of the radioactivity is released into the supernatant (Wigzell, 1965; Hirschberg et al., 1977). Tests can be made more specific. For example, the effect of a chemical on the beating of cultured heart cells can be monitored by the induction of arrhythmia. The use of cultured kidney tubule cells not only allows assessment of toxicity but enables studies to be performed of the effect of a chemical on membrane transport (Dogani, 1984).

1.4. Animal cell biotechnology The exploitation for commercial purposes of animal cells in culture is only in its infancy and the techniques of mass cell culture are still not perfected (see Chapter 3). It was recognised very early that cells in culture provided an ideal host for growth of viruses and more recently cultured cells have been used in the production of vaccines, monoclonal antibodies, hormones and various other compounds such as interferon, plasminogen activator and blood clotting factors (Butler, 1987; Spier, 1987). Plant cells in culture are also used to produce a wide variety of rare chemicals. The animal cell has a major advantage over the bacterial cell as the medium for expression of cloned mammalian genes. The transcript produced is processed and the protein produced is modified in the correct manner. This is seldom the case for mammalian genes expressed in bacteria. A major problem, however, is that yields are low and very large numbers of cells are required e.g. only 20 pg of interferon-p are produced per litre of medium from human embryonic lung fibroblasts in roller bottles and purification of such small amounts of protein is very difficult (Cartwright, 1987). We should not underestimate the potential of animal cells to multiply, however. A minute biopsy of 1 mm3 will yield lo6 cells which over 50 generations (a typical life span for untransformed cells) will multiply to give lo2’ cells: equivalent to the biomass of 10 million people (Butler, 1987).

CH. 1 .

INTRODUCTION

9

Interferon is produced in virally infected cells and induces in other cells an antiviral state resulting at least in part from a specific inhibition of viral mRNA translation (Revel and Groner, 1978). Interferon shows host cell specificity and therefore production of human interferon must be performed with human cells or using the human genes expressed in bacteria. Fibroblasts and leukocytes have been used and the inducing viruses include Sendai and Newcastle Disease Virus (Gresser, 1961; Baron and Isaacs, 1962; Merigan et al., 1966). In addition, homologous cell cultures must be used in the assay of interferon and primary late embryo cultures or foreskin fibroblasts have been used (Merigan et al., 1966). The diploid cell line WI38 is less sensitive. The assay for interferon involves incubating cells overnight with increasing dilutions of interferon and then challenging the cells with, say, vesicular stomatitis virus (VSV) at 20 p.f.u. per cell. Twenty hours later the culture fluids are harvested and assayed for VSV using a plaque assay (8 14.3.1) on mouse cells. The greatest dilution of interferon which inhibits virus yield by 3.2 fold (0.5 log,,) contains 1 unit of interferon (Baron, 1969). The aim of most technologies is to generate a population of cells each of which secretes a desired product into the medium. For this to occur the cells do not need to be actively dividing and, in fact, this is sometimes counterproductive. Cells can remain viable at high cell densities using perfusion systems (Chapter 3) and the product purified from the spent medium (Spier, 1988). Furthermore, the simpler the growth medium (i.e. the fewer protein factors required) then the easier is the downstream processing and this is one reason for the development of serum-free media especially for the culture of hybridoma cells used for monoclonal antibody production (0 5.8). As well as being used as producers of chemicals, cells themselves can be cultured and then reintroduced into the animal in an attempt to cure a disease or repair tissue damage. For instance lymphocytes, stimulated to grow in vitro, can be used to improve the immune response to tumours. One of the fears associated with the therapeutic use of cultured animal cells or products derived from them is that they may be contaminated with material (e.g. DNA) that might produce tumours

10

CELL CULTURE FOR BIOCHEMISTS

in the recipient. Although this fear may have been exaggerated (Butler 1987) it led to the decision only to use untransformed cells for the production of material for introduction into humans (Reaveny, 1985). The commercial exploitation of cell cultures can be gauged from the use of foetal calf serum. Even though this is increasingly being reduced as a component of cell culture media, sales increased more than 6-fold between 1984 and 1987 (Spier, 1987). The value of the market for products made from cultured animal cells has been estimated at 23 billion dollars for 1991 (Ratafia, 1987). These figures underline the importance of animal cell culture as an industrial as well as a research tool.

CHAPTER 2

Characteristics of cultured cells 2.1. Types of cells Cultured cells are described as being fibroblast-like (spindle shaped) or epithelial-like (polygonal). These names are not very useful as the shape of cells varies depending on the medium and cell density. Furthermore, as cells from an increasingly varied tissue source are being cultured, cells are best described with regard to their origin, e.g. neuronal cells, myoblasts, lymphocytes etc. Many tissues contain two or more types of cell and this can lead to confusion. Fibroblasts live in the spaces between other cells and secrete the proteins of the extracellular matrix, e.g. collagen. They do not associate tightly with one another or with other cell types, but they do readily attach themselves to a substratum. In dilute culture they are observed as individual spindle shaped cells which move around the surface of the culture vessel avoiding each other. As their density increases they tend to align themselves in parallel assays (Fig. 2.1). However, fibroblasts can become chondrocytes or adipocytes in the appropriate environment (Taylor and Jones, 1979) and on transformation (8 2.2) they readily lose their contact inhibition and pile up on top of one another (Fig. 2.1) Epithelial cells are cells covering the body surface and bounding cavities, e.g. the gut or kidney tubules. They are bound together laterally by tight junctions to form sheets of cells and their apical and basolateral surfaces differ in composition and are kept apart by the tight junctions. They have the ability to transport solutes across the cell sheet from the apical surface. Of course, in culture a single epithelial cell cannot exhibit these properties but as the cells divide they form stable clusters of tightly associated cells and monolayers of such cells do show polarity. This is most easily achieved in serum-free, hormonally defined medium (5 5.8) when differentiated 11

12

CELL CULTURE FOR BIOCHEMISTS

Fig. 2.1. Untransformed (1) and Polyoma virus transformed (2) BHK cells show quite different morphology. This is also clear in the appearance of the colonies shown at low power (3) where the transformed colony is compact whereas untransformed cells tend to spread out over the culture surface in parallel arrays. (Reproduced from MacPherson and Stoker, 1962, with permission.)

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

13

characteristics are more readily maintained and fibroblast overgrowth prevented (§ 5.8) (Taub, 1985). Thus monolayers of the canine or pig kidney tubule cell lines (MDCK and LLC-PK1) can be induced to transport solutes down through the monolayer causing it to lift off the substratum and form a dome. Inducers of dome formation are similar to those that cause induction of differentiation in erythroleukaemia cells, e.g. DMSO, HMBA, butyrate (see 9 15.1) (Barnes, 1985). Receptors for hormones (e.g. vasopressin, glucagon, noradrenalin) are usually found on the apical surface of epithelial cells and such cells have been much used in the study of hormone action (Taub, 1985). Fibroblasts, along with cells from adipose tissue, cardiac and skeletal muscle and the vascular endothelium, are derived from the mesenchyme whle epithelial cells are obtained from the cervix, kidney tubes, bronchi, trachea etc. Neurones and glial cells are of neuroectodermal origin and blood cells arise from the haemopoietic system. The mesenchymal component of many tissues secretes the basement membrane upon which the epithelial component rests; or it induces the epithelial component to secrete its own matrix or basement membrane. For this reason some cells will only maintain their differentiated characteristics when cultivated with fibroblasts or when grown on a surface of collagen or gelatin (denatured collagen). Under such conditions endothelial cells from the lining of blood vessels will develop internal vacuoles and, in the presence of tumour angiogenesis factor, will join up to form a network of capillaries after 3 weeks in culture (Folkman and Haudenschild, 1980; Gospodasowicz et al., 1979).

2.2. Primary cells and transformation Cells taken from an animal and placed in culture are termed primary cells until they are subcultured (Chapter 4). Primary cells, if successfully established in culture, will multiply and will require regular subculturing. However, specialised or terminally differenti-

14

CELL CULTURE FOR BIOCHEMISTS

ated cells often fail to grow in culture unless specifically stimulated by treatment with mitogenic agents (6 2.5). On reaching confluence a primary culture may be subcultured into two or four new bottles and this subculturing may be repeated at about weekly intervals for several months. In such a culture the cells may remain diploid and retain many characteristics of the initial explant. This is a cell line, and several different lunds of cells may be present and some of the characteristics may prove unstable. The cells may be cloned (Chapter 7) and some clones may exhibit a stable phenotype. Such is the WI 38 cell strain, a commercially available strain of human embryonic lung cells, many identical cultures of which were frozen after only a few passages. Early investigators believed that somatic cells would proliferate indefinitely in culture in an unmodified form if they could be maintained in suitable conditions. However, it is now realised that this is not so. Primary cells are readily established from many tissues and for a while these will proliferate exponentially, but after about 6 months the growth rate falls and by 10 months the cells degenerate and die. This takes place after some 50 generations when about cells have been produced in culture from each initial primary cell (Hayflick and Moorhead, 1961). (Although primary cultures derived from embryonic cells will grow for about 50 generations, cells from adult tissue usually enter senescence after about 20 generations.) During the early stages the cells remain euploid (i.e. have the correct diploid complement of chromosomes) but later they become aneuploid. Associated with this cell ageing process is a steady decrease in the level of methylcytosine in the DNA (Wilson and Jones, 1983). This may result in a failure to correctly package the DNA and with aberrant gene expression (Adams, 1990). Very occasionally, one of these aneuploid cells will survive and continue to grow, and it is this phenomenon which has led to the origin of the established cell strains which show the property of immortality. The frequency of this transformation event can be increased by treatment of cells with mutagens - e.g. methylcholanthrene treatment led to the isolation of the L strain of mouse cells (Earle, 1943) from which L929 cells were later cloned - or with some viruses (see Chapter 14). The chromosomal pattern of these cells is markedly

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

15

aneuploid and variable. Thus the chromosome complement of L929 cells varies between 56 and 241 with a mode of 66. Morphological alterations also occur and the L929 strain shows little resemblance to the initial fibroblasts from which the cells were derived. There are several stages to the transformation process. Some cell strains (e.g. mouse 3T3 cells) have gained only the property of immortality, whereas in others this is accompanied by an increased rate of cell division and a decrease in contact inhibition reflected in the ability to grow to high cell densities. NIH 3T3 cells undergo further spontaneous transformation at a rate which depends on their recent history (Rubin and Xu, 1989). Thus late passage cells subcultured into a nutritionally poor medium show very numerous foci representing clones of cells with reduced contact inhibition. Such cells often show reduced nutritional requirements relative to primary cells. This is the situation with BHK 21 hamster fibroblasts (Macpherson and Stoker, 1962; Stoker and Macpherson, 1964) which still have the correct diploid chromosome number but which are believed to have the incorrect chromosome complement. These cells do, however, exhibit a certain amount of contact inhibition of movement (0 2.4.2) and may be further transformed by treatment with polyoma virus (e.g. to form Py Y cells; Fig. 2.1) or SV40 (to form SV28 cells). Highly transformed cells no longer require to grow attached to a substratum (9 2.4) and can be cultivated in soft agar. Such cells will often form tumours when injected into suitable animal hosts. The various stages of transformation are associated firstly with a decreased spreading of cells on substrata and eventually by growth in the complete absence of attachment (Vasiliev, 1985). Thus changes in the extracellular matrix and cytoskeleton which reduce spreading and cause the cells to assume a spherical shape are accompanied by changes which allow such cells to grow. These latter changes result from increased production of, or sensitivity to, growth factors which are discussed further in 0 2.5. HeLa cells (Gey, 1955) were derived originally from tumour tissues and appear to have been transformed in vivo. Although not all neoplastic cell populations will grow indefinitely in vitro many of the human cell lines in common use have such an origin (e.g. HEP2, KB, Detroit 6).

16

CELL CULTURE FOR BIOCHEMISTS

It is important to reahse that there is an alternative explanation to the appearance in a primary culture of rapidly growing ‘transformed’ cells of different karyotype and morphology, i.e. the primary cells may have become contaminated by a cell line being carried in the same laboratory. This happened in Parker’s laboratory when L929 cells contaminated a series of primary cultures and subsequently outgrew the primary cells. Only after careful karyotypic analysis and transplantation specificity tests was this confirmed (Parker, 1959). Concern over a repeat occurrence has led to stringent worlung routines, but cross-contamination may still occur especially in laboratories (even commercial laboratories) where many cell lines are routinely passaged. Lavappa (1978) has shown that 21 cell lines held by the American Type Culture Collection are derivatives of HeLa. Standard immunological and virus susceptibility tests are now available for species identification. It is important to monitor cells to establish that they continue to show the properties originally present. The genetic origin can be checked by karyotyping (0 7.4), DNA fingerprinting (Jeffreys, 1987) or isoenzyme analysis. Using antibodies to different intermediate filaments will indicate the type of cell present (e.g. vimentin is characteristic of mesenchymal cells and desmin of muscle cells). The relationship between generation number and passage number depends on the split ratio. Thus if cells are split so that the contents of one bottle are distributed between two new bottles (split ratio of 1 to 2) then passage number and generation are the same. This is because the cells will only be able to double in number before they again achieve confluence and require subculturing. However, with a split ratio of 1 to 4 the age of the cells in generations will be twice the passage number.

2.3. Growth control 2.3.I . Nutritional requirements

In the 1940’s and early 1950’s most cells were grown in plasma or fibrinogen clots, in the presence of tissue extracts and their ultra-

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

17

filtrates. Two cell lines, the mouse L cell (Sandford et al., 1948) and the HeLa cell (Gey, 1955), were cultured on the surface of glass containers, and in a classical paper Eagle (1955) investigated their nutritional requirements. He was able to propagate these cell lines in the presence of a defined mixture of amino acids, vitamins, salts and carbohydrate supplemented with a small amount of dialysed horse or human serum. Specific nutritional deficiencies were produced by omission from the medium of particular amino acids or vitamins and these could be ‘cured’ by restoration of the missing component. Twenty-seven factors were defined as essential for growth in the presence of a serum supplement and they formed the basis of a medium known as ‘basal medium, Eagle’ or BME. Thirteen amino acids were essential, the remaining six non-essential amino acids being synthesised from other carbon sources. Omission of any one of seven vitamins led to the development of deficiency symptoms. Thus Eagle by using cell culture techniques was able to demonstrate the nutritional requirements of mouse and human cells. This basal medium required frequent replenishment for the cells to continue growing and was shortly replaced by Eagle’s minimum essential medium (MEM) (Chapter 5 and Appendix 1) in which the concentrations of the various components are increased to enable cells to continue growing in culture for several days between medium changes. One of the problems that arose when attempts were made to grow single cells in culture or cells at low density (e.g. 100 cells/ml) was that all of the cells died or grew only very slowly. Cells were able to grow at low density if, in addition to the normal requirements, serine (Lockart and Eagle, 1959) or cystine (Eagle et al., 1961) were supplied. These and other population dependent requirements were investigated by Eagle and Piez (1962) and this led to the concept of ‘conditioned medium’ (but see below). Although cells are able to synthesise these additional requirements they are lost from the cell to the environment in amounts which exceed the biosynthetic capacity of the cell. Conditioned medium is medium in which the concentration of metabolites has built up to such a level that an equilibrium is achieved between metabolites lost from the cell to the medium and metabolites taken up from the medium by the cells.

18

CELL CULTURE FOR BIOCHEMISTS

2.3.2. Cell cycle and growth cycle Growing cells undergo regular divisions about once every 24 h. In between divisions (i.e. during interphase) they double their complement of DNA during a distinct period known as the DNA synthetic or S-phase. S-phase is separated from cell division or mitosis (M) by two gaps (G1 and G2) (see Fig. 10.1). Cells which are not restricted in any way will proceed indefinitely around this cycle (the cell cycle) and are said to be in exponential growth as the cell number doubles on each circuit. Normally, after a short period of exponential growth, some factor becomes limiting. Possibly the area for growth is completely covered or some factor in the medium becomes exhausted. This can be readily achieved by maintaining cells in medium containing 0.5% serum. The rate of growth of the culture then slows down and the size of the culture reaches a plateau (the terminal cell density). When no further cell division occurs the cells are in a quiescent or stationary phase. When the limiting factor is restored such cells reenter the cell cycle in the G1 phase and undergo a round of DNA synthesis prior to cell division. Much effort is going into the search for the factors in serum which generate this mitogenic response (see 8 2.5). There are two or more theories which attempt to explain this type of growth control (6 10.4) but they all propose a point of control shortly after cell division. Once t h s point has been passed a cell will proceed around the cell cycle and divide. As most of the cells in an animal are under some form of growth control they are not proceeding around the cell cycle but have been arrested shortly after mitosis. Thus on setting up a culture of primary cells the restrictions on growth must be removed before those cells can proceed towards division. The cells in some rapidly growing tumours appear to have lost their susceptibility to growth control (see 0 2.5.4) and for this reason primary cells obtained from tumours may be readily established in culture and may grow to higher densities than cells from normal tissues (see below). Giant cells sometimes arise in cultures especially if the growth conditions are not optimal. They are produced by the failure of growing cells to divide and they may increase in size until they are 1

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

19

mm or more in diameter. The incidence of such cells is markedly increased by irradiation (Tolmach and Marcus, 1960). The presence of an occasional relatively small giant cell in a population probably represents no threat to biochemical experimentation, but if their incidence rises it is a reflection of poor culture conditions; such cultures should be discarded and fresh ones obtained and grown in improved media.

2.4. Attachment and spreading 2.4.1. Anchorage dependence and growth in suspension

Although lymphocytes show no tendency to aggregate in vivo and will grow in suspension in vitro (0 6.2) most mammalian cells both in vivo and in vitro grow attached to a substratum either of other cells, of collagen, or of glass or plastic (Klebe, 1974). These two-dimensional substrates (glass or plastic) are very unphysiological, but they offer the advantage to the biochemist that the cells are readily observed and handled. Plastic surfaces need to be specially treated before cells will attach and eukaryotic cells will not attach to bacterial plastic dishes. When buying plastic ware it is important to specify that it is for tissue culture; the letters TC frequently appear in the catalogue number. As cells grow they deposit an extracellular matrix containing collagen, glycosaminoglycan, proteoglycan and glycoprotein (Hay, 1981). Collagen is the major factor, and plays a role in the determination of cell shape, adhesiveness and proliferation and differentiative potential (Barnes, 1984). Klebe (1974) describes the preparation of collagen-coated dishes. Another favourite substratum for studying anchorage dependence is gelatin (denatured collagen). Dishes may be treated with an aqueous gelatin solution (1% for 2 h at 4OC) and then washed with water and stored at room temperature until required. Cell attachment and cell spreading are two related events but they are not necessarily controlled by the same factors. Attachment of cells to other cells or to a substrate occurs rapidly and may depend

20

CELL CULTURE FOR BIOCHEMISTS

largely on charge. It is not an energy requiring process and is inhibited by serum factors (Walther et al., 1976; Curtis, 1987; Himes and Hu, 1987). Within 2 min at 27°C a bond is formed between cells which is susceptible to 0.01% trypsin. This bond is only formed between cells and not with the substratum. Only after 8 min is a more stable bond formed, probably involving serum spreading factors such as fibronectin. Spreading follows in response to signals passed through the membrane to the cytoskeleton (0 2.4.3). Although cells growing attached to a substratum have advantages in some systems, for other purposes a suspension culture may be preferable. In general, however, those cells dislodged from a substratum on which they are growing fail to grow in suspension and quickly degenerate. Earle et al. (1954) showed that if L cells were maintained in a roller bottle rotating at 40 r.p.m. they failed to attach to the surface and that the addition of methylcellulose (Methocel) at 0.1% prevented clumping and maintained viability. A number of cell strains have now been selected which grow readily in suspension, e.g. HeLa S3 and LS cells, while other cell strains, e.g. A9, will grow either attached to the substratum or in suspension depending on the nature of the salt solution in which they are grown, i.e. omission of divalent ions and an increase in the phosphate concentration favours growth of cells in suspension (see 8 5.2 and Eagle, 1959). 2,4.2. Density dependent regulation (contact inhibition)

Primary cells will continue or start to divide in culture but exhibit contact inhibition of movement (Abercrombie and Heaysman, 1954). When two such cells approach one another the characteristic ruffling movements of the cell membrane stop in the area of contact. Primary cells therefore do not grow one on top of the other and, in general, cease to divide when a monolayer has been formed. This phenomenon is not restricted to primary cells but applies also to many cell lines. An ideal example is the 3T3 mouse fibroblast cell line which grows rapidly in sparse culture but all division stops as soon as the cells become confluent at about lo6 cells per 6 cm dish (Holley and Kiernan, 1968). Such cells may for some time remain

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

21

healthy in this quiescent state. The actual cell density is related to the concentration of serum in the medium and Todaro et al. (1965) have shown that addition of serum to an inhibited culture results in a round of DNA synthesis and cell division. A number of factors have been isolated from serum which show some ability to overcome contact inhibition (see 6 2.5) and cells transformed by viruses (0 14.5) show reduced contact inhibition (Holley and Kiernan, 1968; Dulbecco, 1970) and grow to a higher terminal cell density. They are said to have lost density dependent regulation. Transformed cells, in contrast to untransformed cells, continue to grow until they have exhausted the medium, and unless this is quickly replenished such cells soon die. It would seem that the growth of transformed cells is less dependent on the macromolecular components of serum (i.e. hormones or growth factors: 0 2.5) and becomes limited only when some of the low molecular weight nutrients become exhausted. As cells in a monolayer grow two changes occur: (1) the cells become more crowded and less flattened and so expose a diminished surface area to the medium; (2) the medium becomes depleted in nutrients etc., especially in a zone immediately surrounding the cells (Stoker, 1973). If a strip of cells is removed from a confluent monolayer of untransformed cells (e.g. 3T3 mouse embryo cells) then the cells at the edge of the wound are stimulated to synthesise DNA and divide. They quickly colonise the unoccupied area of the wound. This phenomenon known as top0 inhibition (Dulbecco, 1970) is now explained by the presence in cells on the edge of the wound of an increased surface area exposed to the medium (i.e. neighbouring cells have been removed) (Stoker, 1973; Dulbecco and Elkington, 1973). The fact that crowded cells are less flattened and that cells in suspension are spherical and fail to grow, was followed up by Folkman and Moscona (1978) who showed that the extent of cell spreading was closely related to growth in non-transformed cells (Vasiliev, 1985). 2.4.3. Fibronectin

0 2.5 describes the growth factors (hormones) which need to be present in serum or media supplements for cell growth. In addition

22

CELL CULTURE FOR BIOCHEMISTS

attachment factors responsible for cell :cell and cell :substrate interaction must be supplied. Fibronectin is the major attachment factor and it also promotes cell :cell adhesion and flattening of cells on a glass or plastic surface (Yamada and Olden, 1978; Olden et al., 1979, Yamada et al., 1982). Fibronectin is the major cell surface glycoprotein and is also known as CIG (cold insoluble globulin), CSP (cell surface protein), 2-SB (surface binding glycoprotein) and LETS (large external, transformation-sensitive) protein. It has a molecular weight of 220,000 but exists as disulphide linked dimers or higher oligomers. Fibronectin is found, in related forms, in serum as well as on the surface of normal, but not transformed cells. Addition of fibronectin to transformed cells causes a partial return to the normal phenotype in that it increases the adhesion of cells to other cells and to the substratum. It will also increase the saturation density achieved by 3T3 cells (Yamada et al., 1982). In contrast, antibodies to fibronectin will induce some of the characteristics of transformed cells in otherwise normal cells. Fibronectin is not present on mitotic cells and increases in amount on normal cells as they reach confluency; or on cells arrested by low serum treatment (8 2.3 and 11.6) (Pearlstein, 1976; Hynes and Bye, 1974). Fibronectin is a very flexible molecule consisting of several loosely linked domains (Alexander et al., 1978; Yamada et al., 1982) and forms a relatively immobile fibrillar network on the cell surface from which it can be removed by treatment with very low levels of trypsin (Pearlstein, 1976) or by 1 M urea, for which reason it was realised not be be an integral membrane protein. Along with other extracellular matrix proteins such as laminin, vitronectin (serum spreading factor) and collagens, it interacts with the cell membrane via specific receptors (Pytela et al., 1985). This receptor binding is dependent on the presence of divalent cations such as Mg2+ or Ca2+ (Edwards et al., 1987). Binding to the receptor is followed by cytoskeletal changes leading to cell spreading (Hynes, 1981; Vasiliev, 1985).

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

23

2.5. Growth factors 2.5.1. The serum component

The undefined factor in Eagle’s media is the serum components. It used to be fashionable to dismiss the serum requirement for cell growth by saying that serum contains various undefined components essential for cell attachement and cell division. However, many of these components have now been defined and this section will give an indication as to the nature of some of the mitogenic peptide growth factors and how they act. As described in 8 2.3 and 2.4, these are not the only factors in serum, which also provides essential inorganic components, cell attachment factors and a variety of compounds required to maintain differentiated functions and which may limit the growth of particular cell types. The factors which affect cells may be produced by the same cell as responds to them (an autocrine response shown by some tumour cells), or may be produced by a neighbouring cell type (a paracrine response mediated by the interleukins). It is sometimes only possible to distinguish autocrine and paracrine responses by cloning cells from a particular tissue, as a factor produced by one cell type may be ‘processed’ by a second cell type before reacting with receptors on the first cell type. Cell growth factors may also be produced by a distant cell or tissue and travel to the responding cell via the blood stream (an endocrine response as shown by insulin). These factors are classically known as hormones. Although endocrine factors are present in serum, conditioned medium is frequently a better source of autocrine and paracrine factors. One of the earliest factors to be discovered was platelet derived growth factor (PDGF). This arose from the finding that plasma obtained from unclotted blood could not replace serum in promoting cell growth. PDGF was purified from serum and shown to be the component missing in plasma. A large number of peptide growth factors have now been isolated (Table 2.1) and they tend to fall into families exhibiting partial

24

CELL CULTURE FOR BIOCHEMISTS

TABLE 2.1 Growth factors A. Insulin-like growth factors - derived from one polypeptide of 6-12 kDa mitogenic Insulin: only mildly mitogenic IGF-1: (somatomedin C) present in plasma and produced by human liver fibroblasts on stimulation by PDGF or GH IGF-2 (somatomedin A) - similar to IGF-1 MSA (multiplication stimulating activity) - the rat equivalent of IGF-2. Present in conditioned medium from rat liver (BRL) cells. NGF (nerve growth factor) - required for maintenance of neuronal differentiation. Obtained from human melanoma cell line A375.

B. Cell-derived growth factors - present as dimers of polypeptides of 17-30 kDa. Mitogenic factors acting on mesenchymal cells, e.g. fibroblasts, glial cells, muscle cells Derived from platelets (PDGF), fibroblasts (FDGF), glioma cells (GDGF), eyes (EDGF - related to acidic FGF) and neural tissue (endothelial cell growth factors - ECGF-a and 8) etc. 8-ECGF is related to a-FGF by loss of 14 N-terminal amino acids and to loss of 20 N-terminal amino acids C. Epidermal Growth Factors (EGF) and Transforming Growth Factor-a (TGF-a) present as single polypeptides of 6-7 kDa and bind to a common receptor. EGF (P-urogastrone) is present in urine and submaxillary glands and TGF is obtained from conditioned medium from transformed cells. Act on mesodermal and ectodermal cells EGF is usually mitogenic (but sometimes inhihitory) and TGF-a is mitogenic and allow cells to grow in soft agar Sarcoma Growth Factor (SGF) - obtained from sarcoma virus transformed murine 3T3 or rat kidney cells is probably related D. Transforming Growth Factor-8 - present as 2 chains of 24 kDa. Isolated along with TGF-a and may be mitogenic or inhibitory to cell growth. Transforms cells in present of TGF-a.

E. Colony-Stimulating Factors - 24-35 kDa glycoproteins produced by interstitial cells of most tissues (except Multi CSF) but not present in serum. Have dual functions of causing growth and differentiation of bone marrow cells Macrophage CSF - stimulates macrophage production Granulocyte CSF (P-CSF) stimulates granulocyte production GMCSF (a-CSF) - stimulates production of macrophages and granulocytes Multi CSF - stimulates production of most myeloid cells only by activated T-cells and myeloid leukaemia cells Erythropoietin - produced in liver and kidney and stimulates growth and differentiation of erythrocyte precursors

CH. 2

CHARACTERISTICS OF CULTURED CELLS

25

TABLE2.1 (CONTINUED)

F. Interleukins - peptides which transfer signals between white blood cells. In this they are helped by interferons (which are growth inhibitors), tumour necrosis factor (TNF),the CSFs and TGF-8. Interleukin 1 (a and 8) shows sequence similarity to F G F and stimulates 11-2 production and activates B-cells Interleukin 2 (T-cell growth factor) is produced along with IFN-.I by activated T-lymphocytes and stimulates growth and differentiation of B-lymphocytes and macrophages Interleukin 3 is multi CSF The table is based on material found in: Nissley et al. (1979), Todaro et al. (1979), Ross et al. (1982). Anzano et al. (1983), Heldin and Westermark (1984), James and Bradshaw (1984), Barnes (1984), Deuel (1987), Nicola and Vadas (1988), Sporn and Roberts (1988), Akhurst et al. (1988), Derynck (1988) and Old (1988). Further information can be found in Growth Factors: Structure and Function (1985) C.R. Hopluns and R.C. Hughes, eds, J. Cell Science, Suppl 3. See also Table 2.2.

homology at the amino acid level and interaction with common receptors (see 0 2.5.3). The somatomedins, which in vivo mediate the action of growth hormone, are related to insulin and have been renamed insulin-like growth factor 1 and 2. Their mitogenic effect is, however, very much greater than that of insulin. PGDF is related to at least two other factors derived from transformed fibroblasts (FDGF) and ghoma cells (GDGF). All three have a mitogenic effect on cells of mesenchymal origin e.g. fibroblasts, ghal cells or muscle cells. Factors such as PDGF are required to maintain cell division and are necessary to induce a non-dividing cell to enter the cell cycle (see 0 2.5.2). PDGF will not, however, override the growth controls shown by normal cells, i.e. it leaves the cells in a state where growth is still controlled by space limitations and in which the cells still require to be attached to a suitable substratum, i.e. do not grow in soft agar. A second type of growth factor (e.g. transforming growth factor+ (TGF-P), sarcoma growth factor) will induce the transformed phenotype in which cells will grow on top of one another reaching very high cell densities and will grow in soft agar. TGF-P is a member of a family of peptide factors which also show a variety of

26

CELL CULTURE FOR BIOCHEMISTS

biological activities, regulating adipogenesis, myogenesis, osteogenis and epithelial differentiation. It is believed it acts by altering the architecture of extracellular matrices (Ignotz and Masague, 1986; Masague, 1988). Some factors may be required to maintain differentiated functions whereas others may precipitate into growth previously quiescent cells, or alternatively, be required to preserve limitations on cell growth pertaining in vivo. The striking observation that the fusion of an immortal somatic cell with a mortal cell (i.e. a cell with a limited potential for division) gives rise to mortal cells (Pereira-Smith and Smith, 1983) shows that regulation of growth potential (in the presence of all necessary positive factors) is via a repressive mechanism. Breakdown of this mechanism of programmed cell ageing leads to unlimited growth (Weinberg, 1988). The action of a growth factor may differ depending on the state of differentiation of the target cell or on the presence of other growth factors. Thus in the presence of PDGF, TGF-P stimulates growth of fibroblasts but if EGF is present its effect is inhibitory (Sporin and Roberts, 1988). 2.5.2. Growth factors for haemopoietic cells A particularly large number of factors are known to affect the

growth and development of haemopoietic cells, the many different types of which arise from a single progenitor stem cell. Other systems may be analogous but, as yet, have not been studied in such detail. Most mouse tissues produce three colony stimulating factors (CSF) which are glycoproteins acting on bone marrow cells to stimulate the growth and differentiation of (a) macrophages (MCSF), (b) granulocytes (G-CSF) and (c) macrophages and granulocytes (GM-CSF). A fourth factor (multi-CSF or interleukin-3: 11-3) is produced by activated T-lymphocytes and stimulates production of many different types of blood cell. Interleukin-5 (11-5) is a fifth factor which stimulates production of eosinophils (Hamblin, 1988). The mitogenic action of these factors is distinct from their function in differentiation as can be seen from the observation that G-CSF is required for differentiation of myeloid leukaemia cell lines which do

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

27

not need it for growth. The production of CSFs by many tissues is under the control of interleukin-1 (11-1) and tumour necrosis factor (TNF) which are produced by activated macrophages while M-CSF is produced in respone to 11-3 (Dexter and Spooner, 1987). Erythropoietin is made in the kidney in response to the oxygencarrying capacity of the blood and is required at all stages in the growth and differentiation of red blood cells. The control of production of the various classes of lymphocyte is also under control of interleukins (growth factors which mediate the communication of white blood cells though they may also interact with other-cell types). Thus a T-cell activated by 11-1, TNF, TGF-P or interferon-a (IFN-a) synthesises interleukins 2,3,4,5 and 6, IFN-y and GM-CSF which (1) act to induce growth and differentiation of bone marrow cells and (2) act on B-lymphocytes to cause differentiation and antibody production and (3) induce further production of growth factor by macrophages to stimulate growth and/or differentiation of stem cells, T- and B-lymphocytes and fibroblasts and endothelial cells. Two or more factors may act synergistically or antagonistically on the same cell. This is an exceedingly complicated network (see Table 2.2) whose details are being worked out partly using cultures of purified white blood cells (Golde and Gasson, 1988). Hamblin (1988) has written an introductory text on lymphokines. Lymphokines are secreted into the culture medium by hybridomas (0 13.6) and may be isolated following growth of cells in bioreactors (9 3.8) in serum-free medium (6 5.8.3). This is very tedious, however, and as the various genes are cloned, production in prokaryotes or yeast is becoming more common. 2.5.3. Mechanism of action of peptide growth factors Some growth factors (e.g. PDGF) induce DNA synthesis in quiescent 3T3 cells when no other growth factors are present, while others (e.g. EGF and IGF) are required in combinations indicating that all factors do not act by a common mechanism (Rozengurt, 1986; Wakelam, 1989).

28

CELL CULTURE FOR BIOCHEMISTS

TABLE2 2 Factors produced following ingestion of a bacterium with a lipopolysaccharide coat by a macrophage. Based on data in Old (1988) Factor Produced by Interacts with

IL-1 x IL-2 IL-3 IL-4 IL-5 IL-6 X TNF X TGF-8 X IFN-CX X IFN-8 IFN-1 G-CSF X M-CSF X GM-CSF X PDGF X

x

x

x

x

X X

X

X

x

x

x

X X

X

x

x

x

x

x

X

X

X

X

x

x x x

X

x x x

x x

x x

x

X X

X X

x

x

X

x x x

x x x

X X

x

x

X

On stimulation of quiescent cells with growth factors or serum there is a rapid increase in the transmembrane flux of Na+, K + and H + and a mobilisation of Ca2+ from intracellular stores. The increase in Ca2+ concentration can be mimicked by treating cells with the Ca2+ ionophore, A,,,,,. There quickly follows a series of events leading to changes in gene expression and cell structure and eventually to DNA replication and cell division. Growth factors are active at very low concentrations, i.e. about 1 pM. They exert their effect by binding to specific cell surface receptors with very high affinity. Related factors show lower affinity for the receptors of other members of the group. Following interaction with its receptor, the growth factor is internalised and transported via endocytotic vesicles to the lysosomal compartment where

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

29

breakdown of the peptide growth factor occurs. Although the major action of peptide growth factors is believed to result from the initial interaction with the cell surface receptors (Wakelam, 1989; Wahl et al., 1989a), there is some evidence that there may also be a nuclear site of action similar to that seen for steroid hormone (Rozengurt, 1986; Burwen and Jones, 1987; see 9 2.5.5). The receptors for growth factors are membrane glycoproteins present at between 5000 and 500,000 molecules per cell. At least two second messenger systems are involved employing either cAMP or diacylglycerol (DAG) and inositol trisphosphate (IP,) (Berridge and Irvine, 1984; Dumont et al., 1989; Wakelam, 1989). Receptors are linked to adenyl cyclase or to phospholipase C via GTP binding proteins (G-proteins, Sagi-Eisenberg, 1989) parts of which resemble the p21 proteins encoded by the rus oncogenes (Marshall, 1984,1987; Wakelam et al., 1986; McCormick, 1989). For example, TSH binds to a receptor which activates adenyl cyclase and the resulting cAMP acts as a second messenger to activate protein kinase A. This phosphorylates several proteins leading to increased expression of c-fos and c-myc and to a mitogenic response (Dumont et al., 1989) (see below). A number of other factors activate phospholipase C which causes the hydrolysis of phosphatidyl inositol(4,5)bisphosphate to DAG and IP,. Following release the IP3 is rapidly broken down via IP, and IP, to inositol in a Li' sensitive reaction and this is the reason why lithium can desensitise receptors. The inositol then reacts with activated DAG and is rephosphorylated to regenerate phosphatidyl inositol(4,5)bisphosphate (Berridge and Irvine, 1984; Wakelam, 1989). Phospholipase C can also be activated directly by means of the tyrosine kinase activity of the receptor (James and Bradshaw, 1984; Deuel, 1987; Massague, 1987). Thus on binding EGF, the EGF receptor acts to phosphorylate certain tyrosines on a membrane bound phospholipase C-I1 (C-y) (Wahl et al., 1989a; Margolis et al., 1989; Meisenhelder et al., 1989) and the PDGF receptor acts in a similar manner (Wahl et al., 1989b; Meisenhelder et al., 1989). The tyrosine kinase activity associated with the G F receptor can phosphorylate G-proteins (Zick et al., 1987).

30

CELL CULTURE FOR BIOCHEMISTS

IP, acts as the second messenger for Ca2+ mobilisation from the endoplasmic reticulum while DAG stimulates the Ca2+ sensitive protein kinase C (which modulates EGF receptor) and activates the plasma membrane Na+/H+ exchange carrier. In this function DAG is mimicked by phorbol esters which have a tumour promoting function. The effect of stimulating Na+/H+ exchange is to increase the intracellular pH and K + concentration, conditions which are necessary to induce or maintain the proliferative response in quiescent cells (Rozengurt, 1986; Wakelam, 1989). Rather than being reincorporated into inositol phospholipids, DAG can be broken down to release some arachidonate which is the precursor of prostaglandin E which, in turn, can react with plasma membrane receptors linked to adenyl cyclase and CAMPproduction. IL-3 does not activate phospholipase C but does promote phosphorylation of the glucose transporter by phosphokinase C (Dexter and Spooner, 1987). Within 5-20 min of stimulation of quiescent cells with PDGF or EGF there occur transient increases in the nuclear proteins coded for by the oncogenes c-fos c-jun and c-myc (Lau and Nathans, 1987; Sassone-Corsi et al., 1988; Chiu et al., 1985; Robinson, 1988; Nathaus, 1987; Ryseck et al., 1988; Lamph et al., 1988; Quantin and Breathnach, 1988). Growth factors exert their effect on gene expression through the mediation of cis acting 'serum responsive elements' (SRE) present in the enhancer region of several such genes. A serum responsive factor (SRF) is always bound to the SRE but transcription is inhibited by a labile repressor (Subramaniam et al., 1989; Herrera et al., 1989) and the effect of serum factors is potentiated by treatment with cycloheximide. Protein kinase C also modulates the activity of the transcription factor AP-1 thereby activating transcription of a number of genes including that for collagenase and the proto-oncogenes c-fos, c-myc and c-sis (Spandidos et al., 1988). c-fos proteins etc. are part of, or act on, transcription factors which in their turn play a part in controlling the changes in gene expression required for the mitogenic response (Marshall, 1987). Later responses are the increased expression of the c-myb and p53 genes in late G1 (Reich and Levine, 1984; Thompson et al., 1986) followed by the production of the various

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

31

proteins required for DNA synthesis and cell division (see Chapter 10). 2.5.4. Oncogenes

Mutation may alter the amount or nature of a controlling growth factor or its receptor either directly or indirectly, thereby affecting the immortality of a cell. In addition, transforming viruses may introduce into recipient cells, genes (viral oncogenes) coding for proteins whch mimic growth factors or affect their production or activity. For example the sis gene of simian sarcoma virus codes for a protein virtually identical to part of PDGF (Waterfield et al., 1983) and the src gene of Rous sarcoma virus codes for a tyrosine kinase which, like the cell surface receptors for EGF, PDGF and IGF can phosphorylate intracellular proteins involved in the transduction of the growth signal to the nucleus (Cooper and Hunter, 1981). A major substrate for tyrosine kinase is cdc 2 protein kinase - the best characterised cell cycle regulator in eukaryotic cells (Draetta et al., 1988) (see 5 10.4). Permanent upregulation of genes encoding receptors (erb A for thyroid hormone receptor, erb B for EGF receptor) or components of the GTP-binding protein complex (rus) or nuclear transcription complexes can also lead to loss of growth control although it is at least a two stage process to pass through immortality via uncontrolled growth to full tumourogenicity (Spandidos and Wilkie, 1984; Hunter, 1984; Marshall, 1987; Katan and Parker, 1988; Schwab et al., 1985; Sporn and Roberts, 1985; Heldin and Westermark, 1984). The EIA protein of adenovirus binds to and inactivates the product of the retinoblastoma (RB) gene - an antioncogene. The RB protein in some way acts to control cell proliferation and its inactivation has been implicated in a number of different human tumours (Whte et al., 1988; Weinberg, 1988). Tumourogenicity can also be induced by overproduction and mutation of the p53 protein which appears to be an antioncogene product s i d a r to that of the RB gene (Finlay et al., 1989). The altered p53 protein binds tightly to the transforming proteins of

32

CELL CULTURE FOR BIOCHEMISTS

SV40 and adenovirus and this may indicate how they exert their effect (Eliyahu et al., 1985; Lane, 1984; Wang et al., 1989). 2.5.5. Steroid hormones

As well as providing peptide growth factors, serum also supplies steroid hormones. These also may act by promoting cell growth and/or differentiation but their mechanism of action is fundamentally different from that of the peptide growth factors. They are transported around the body in the blood stream where they are often found associated with.the albumin fraction. It is perhaps for this reason that albumin appears to be a good growth promoter for some cells. In order to study the action of steroid hormones on cells it is essential that the serum proteins in the culture medium are first stripped of associated hormones by stimng with dextran-coated charcoal (see 0 5.7.1). Being lipid soluble, steroid hormones readily enter cells to interact with their receptors which are present only in the appropriate target cell. These receptors are DNA binding proteins which interact with short recognition sequences in the promoter/enhancer regions of certain genes. When associated with the hormone the receptor activates transcription of these genes. Often the action of steroid hormones can be antagonistic. For instance, oestrogen is mitogenic in endometrial cells but this action is reversed by progesterone. However, the mitogenic effect of oestrogen on the breast cancer cell line MCF-7 is mediated via peptide growth factors and potentiated by progesterone which causes increased production of the EGF receptor. Oestrogen upregulates production of TGF-a which stimulates growth by interacting with the EGF receptor (Leake, 1988).

2.6. Differentiated functions in cell cultures Although in most cases cells derived from primary cultures quickly revert to undifferentiated fibroblasts and epithelial cells, an increasing number of cell lines have been produced which retain some of the functions of the tissue of origin. One of the major problems in

CH. 2.

CHARACTERISTICS OF CULTURED CELLS

33

establishing lines of differentiated cells was the presence of the much faster growing undifferentiated fibroblasts in the initial explant (see Chapter 15 and Fig. 6.2). Normally these fibroblasts rapidly outgrow the differentiated epithelial cells. In some instances, however, the ready attachment of fibroblasts to a substratum has been used to remove them from a mixed culture (Yaffe, 1968; Rheinwald and Green, 1975). Puri and Turner (1978) report that in the absence of serum only fibroblasts from chick muscle tissue adhere to the substratum thus leaving the myoblasts in suspension from where they may be recovered and transferred to a second vessel in serum containing medium (0 15.4). By first converting a solid hepatoma into an ascites cell line, Thompson et al. (1966) were able to establish lines in culture (hepatoma, tissue culture or HTC) which responded to dexamethasone (a steroid hormone analogue) by induction of tyrosine transaminase, i.e. a typical liver cell response. Another method of removing fast growing fibroblasts is to prepare primary cultures from a tumour and after a short time return them to an animal where they will reform a tumour. By repeated alternation of growth in vivo and in vitro it was hoped to select for tumour cells and this has proved successful in isolating differentiated cell lines from adrenal, pituitary and neural tissue (Buonassisi et al., 1962; Augusti-Tocco and Sato, 1969). The loss of differentiated functions is often associated with a high growth rate of the cells in culture and when growth or DNA synthesis is inhibited differentiated functions reappear. Thus a rat pituitary cell liqe, although normally producing growth hormone, will also synthesise prolactin in response to treatment with bromodeoxyuridine at 3 pg/ml (Biswas et al., 1977) and the Friend cells (6 15.1) cease to grow and start to make haemoglobin when treated with dimethylsulphoxide or butyric acid. Such changes probably occur as a consequence of changes in the concentration of various growth factors (see 0 2.5). Some s stems where differentiation may be elicited in vitro are considereJin Chapter 15.Macrophages

This Page Intentionally Left Blank

CHAPTER 3

Culture vessels 3.1. Design of culture vessels For the design of culture vessels the following factors are significant: 1) Do the cells grow in suspension or as a monolayer? 2) The scale of the operation, i.e. are single cells the object of study or are gram quantities required? From the biochemists’ point of view the requirements for their subsequent manipulations must also be taken into account. 3) Is gaseous exchange with the atmosphere allowed or should the vessel be sealed off?

3.1.1. Gaseous exchange Gaseous exchange occurs with Petri dishes and cell culture trays and some types of suspension vessels. Tissue culture flasks often have caps which can be left in a venting position (allowing gaseous exchange) or tightly closed. When the medium used is buffered with bicarbonate (30 mM) it is essential that such cultures be maintained in an atmosphere of about 5% CO, in air. This allows maintenance of the correct pH which is readily monitored by the colour of the phenol red present in the medium. The colour should be a tomato colour - not yellow and not red and certainly not purple indicating a pH of 7.2. Alternatively, media buffered to p H 7.2 with Hepes buffer (20 mM) (see 0 5.2.1) can be used when control of the atmospheric pC0, is unnecessary, In some instances, especially cell cloning. Hepes buffer (20-25 mM) is used in combination with 8 mM bicarbonate when the vessels should be maintained in equilibrium with 2-3% CO,. 35

36

CELL CULTURE FOR BIOCHEMISTS

A variety of CO, incubators are now available which automatically control temperature and pC0,. Some also have an automatic humidity control but usually they operate with a tray of water in the base in an attempt to maintain > 98% relative humidity. The water in the tray must be changed regularly to prevent contamination. Humidity must be maintained high to prevent evaporation of water from the medium in ‘open’ vessels. Such vessels can be obtained with ‘baffles’ or rings in the lids to reduce air circulation. The problem with incubators is the rapid loss of controlled conditions when the door is opened. This is minimised if the circulation fan switches off on opening the door and most incubators have a CO, purge which rapidly restores the CO, level. Some incubators also allow PO, to be controlled but the most imporant factor is temperature, especially for those working with temperature sensitive cells and viruses. Constant temperature throughout the incubator is maintained by fan assisted circulation. However, fans cause vibration and can be the cause of ‘patterning’ in which the cell density varies in a regular way over the bottom of the culture vessel. Another cause of patterning is the holes in the shelf of the incubator. When a cool vessel containing a cell suspension is placed in a warm incubator the flask heats more quickly where it is in contact with the metal of the shelf and this sets up currents within the suspension leading to redistribution of the cells. CO, incubators are available from a number of companies, e.g. Bellco, Forma, Gallenkamp, Grant, Heracus, Leek, Damon/IEC, Napco and Queue (see Appendix 3). 3.1.2. Sealed vessels

These are, for instance, bottles with bungs or tight screw caps and sealed suspension vessels. If the medium is buffered with Hepes no special precautions are required to maintain pH in the early stages of growth. When bicarbonate is the buffer the pH must be maintained by introducing CO, into the vessel prior to sealing. This may be done by passing 5 % CO, in air through the bottle for about 15 sec or by introducing a fixed volume of CO, into the bottle (see Table 3.1 for volumes required, and Fig. 3.1). When changing the medium in a

cn. 3.

37

CULTURE VESSELS

TABLE 3.1 Container

96-well microtitre plate 24-well TC plate 5 cm dish 9 cm dish 60 ml flask 250 ml flask 11 flask (Row) 21 roller bottle 11 suspension flask a

Surface area (Cm2) 0.3 2 19 63 25 15 225 850

Medium required (mu 0.1 0.5 5 10 5 15 50

150 500

Typical cell volume of

Inoculum 11x10~ 5 x lo4 2 x 10’ 1x 106 2 x lo5 6x10’ 2 x 106 1x10’ 1XI08

100% CO, a

-

3 ml 12 ml 50 ml 100ml 50 ml

Volume of CO, required to adjust the atmosphere to 5% CO, in air. This is very difficult to control, and these small flasks should either be gassed with 5% CO, in air, or placed with caps in the venting position, in a CO, incubator until the contents have equilibrated.

Fig. 3.1. CO, delivery systems. On the left is a simple arrangement where air (from an air pump) may be mixed with C 0 2 to produce 5 1 CO, in air for gassing bottles or feeding to an incubator. On the right is an arrangement for measuring out fixed volumes of CO, and delivering them through a three-way tap (at the bottom of the syringe) and a sterile Pasteur pipette to a bottle of cells.

38

CELL CULTURE FOR BIOCHEMISTS

flask of growing cells it is usually unnecessary to gas the flask as the cells ensure the rapid build up of CO,. In contrast, it is preferable to gas a new flask at 10%CO, to ensure that a newly trypsinised, dilute cell suspension gets off to a good start. In both cases the CO, is administered through a sterile plugged Pasteur pipette. The mixture of 5% CO, in air may be obtained directly from a cylinder available from e.g. British Oxygen Company Limited, or may be mixed from air and CO, (obtainable from Distillers Co., Ltd.). Mixers are built into many CO, incubators but are readily constructed from two gas flow meters and an air pump (see Fig. 3.1). 3.1.3. Perfusion techniques

Here gaseous exchange, pH control and medium replenishment occur in a second vessel distinct from the cell growth chamber. From this vessel the medium is pumped into the growth chamber which may be a modified microscope slide, a roller bottle or a capillary bed etc.

3.2. Small scale cultures (Fig.3.2) For those interested in cloning reference should be made to Chapter 7. A small scale perfusion vessel is considered in 0 3.4.3. For many biochemical studies involving incubation of cells with radioisotopes in the presence of drugs, anti-metabolites, hormones etc. small numbers of cells are required and these may conveniently be grown on the bottoms of glass scintillation vials or in the wells of a 6 or 24 well TC plate or even in the wells of a microtitre plate (see Table 3.1). This last method enables 96 replicate cultures to be handled simultaneously but the maximum volume that each well will hold is 0.25 ml. Coverslips are used to greatest advantage when placed in the 24 wells of a tissue culture plate. Each well with its coverslip requires 0.5 ml of medium and can be seeded initially with around 20,000 cells. Alternatively, up to 8 separate cultures can be grown on a

CH. 3.

CULTURE VESSELS

39

single slide using a chamber slide (Lab-Tek, Fig. 3.2a). This is a slide t o which 1-8 culture wells, each of which may be a small as 0.8 cm2, are attached. Cells can be grown in 0.3-0.4 ml medium in the wells whch are later removed leaving the cells attached to the slide for bulk processing. This is a particularly useful technique when testing for mycoplasma contamination (6 9.7). Scintillation vials require 1 ml medium and, although more difficult to handle in large quantities, offer the advantage that at the end of the experiment the labelled cells require fewer manipulations prior to estimation of the extent of radioactive incorporation. Alternatively several coverslips may be placed in a 5 cm plastic Petri dish prior to seeding cells. This has the advantage that each coverslip culture is maintained under identical condition. Coverslips require very thorough washing before cells will grow on them. They should be dropped individually into boiling 0.1 N NaOH or Chloros, then rinsed overnight in running tap water and afterwards given three rinses in distilled water. They should be laid out to dry on clean tissue and sterilised in glass Petri dishes in batches using dry heat. The plastic containers are bought in sterile wraps from commercial suppliers, e.g. Nunc, Falcon, Linbro, Corning, Costar (see Appendix 3). Epithelial cells normally exhibit polarity (Chapter 2) and this is generally not obvious when they are attached to an impermeable plastic surface. Millipore and Costar each produce inserts for 6-well and 24-well tissue culture trays (Fig. 3.2b) which allow the cells to attach to a semi-permeable membrane and to contact the medium on both apical and basolateral surfaces. The medium inside the insert (apical surface) does not mix with the medium in the well outside the insert and Costar’s Transwell is designed to allow sampling of the two compartments with the insert in place. A number of companies supply dishes and TC trays precoated with extracellular matrix collagen, or gelatin (e.g. International Biotechnologies Ltd., and Bibby Science Products Ltd.). This promotes attachment and flattening and is essential for growth of cells such as the F9 teratocarcinoma cells.

CELL CULTURE FOR BIOCHEMISTS

CH. 3.

CULTURE VESSELS

41

Flasks can be readily given a gelatin coat as follows: a) Prepare a 1%gelatin solution in water and sterilise it by autoclaving at 1 5 p s i . for 15 min. b) Dilute the gelatin solution to 0.1% and add sufficient to a flask to cover the growth surface. c) Allow to sit at 4" for 1-2 h . d) Decant the gelatin solution and add the cell suspension in normal growth medium.

3.3. Intermediate scale cultures Cells may be grown in dishes or flasks where the initial inoculum varies from 0.2 X lo6 up to 2 x lo6. The containers may be glass or plastic. The plastic ware is obtained in sterile wraps from commercial suppliers and is specially prepared for use in cell culture (Fig. 3.2a) (see Appendix 3). The glass bottles are usually medical flat bottles but any bottle with a flat side will do provided it is washed correctly and sterilised before use (see Chapter 8). To a large extent the disposable plastic flask has now replaced the glass bottle. Some glass bottles, e.g. Roux bottles, are manufactured especially for growth of cells in culture and these have the advantage of improved optical qualities allowing easier microscopic examination of the growing cells. Some of the larger bottles are known by the name of the person who introduced them.

3.4. Large scale cultures 3.4.1. Roller vessels

These may be 2-2.5 1 Winchester, glass bottles (which are cheap and can be reused) or one of the commercially produced plastic roller Fig. 3.2(a). Vessels in which cells may be cultured. At the top left are three sizes of flask (125, 75 and 25 cm2) and at the bottom left three sizes of dish (9.5 and 3 cm diameter). To the right are shown 6, 12 and 24-well tissue culture trays and a 96-well microtitre plate. At the bottom right is an 8-chamber culture slide. (b) Millicell inserts for 6-well TC trays provide a semi-permeable growth surface.

42

CELL CULTURE FOR BIOCHEMISTS

bottles which have improved optical properties. The cells grow on the inner surface of the bottle and are constantly bathed in medium (150-300 ml/bottle) as the bottle is rotated. The plastic bottles come in a series of lengths (surface area up to 1750 cm’) and Flow Laboratories market a corrugated roller bottle which has a standard length, yet a surface area of about 1750 cm2. Cells cannot be scraped from this bottle and must be removed by trypsinisation. The cost of these plastic roller bottles is high (over E5 each) and they are not generally reusable. It is not, therefore, economical to use them for large scale growth of cells on a regular basis. For such requirements the use of microcarriers should be considered (9 3.4). Machines to rotate the bottles are available commercially from a number of companies (e.g. Wheaton, Luckham, Hotpack, Voss: Appendix 3) and many home made ones are in use (see Fig. 3.3). At Glasgow University one type of machine made in the Biochemistry Department will roll up to 120 bottles and in the Instituto Zooprofilattico Sperimentale in Brescia they can roll up to 7000 bottles simultaneously. The Wheaton roller apparatus comes in modular form. The base unit holds the drive and will roll 5 bottles. Up to 8 additional decks can be added to allow rotation of up to 45 bottles. Also available is an alarm system which gives warning of rotational failure, and an auxiliary battery system which will enable the apparatus to operate for up to 48 h in the absence of an electricity supply. The speed of rotation of the bottles should not exceed 1 r.p.m. and this is too fast for some cells in the initial stages of growth where rotation at 0.25 or 0.5 r.p.m. improves the attachment of cells. One problem that can be encountered with roller bottles is the build up of static electricity which can affect the evenness of cell growth causing bands of densely packed cells to form around the bottles. This is partly overcome if the bottle contacts the rollers only at special ridges which are incorporated one at each end of the modern plastic roller bottle. Sterilin Ltd. (Appendix 3) produce a series of roller bottles containing a spirally wound insert on which the cells grow. These ‘Spira-Cel’ bottles have a growth area of 3000-6000 cm2 but cost

CH. 3.

CULTURE VESSELS

43

Fig. 3.3. Roller bottle machines. This photograph shows three different models. The one at the rear is the Luckham 6-tier model modified by the manufacturer by provision of a more powerful motor. It is very heavy to manoeuvre and only three standard (4:”) Winchester bottles can be accommodated on each tier. In the foreground are two tiers of a stackable model mad by Voss Instruments. Each tier has a separate motor. On the left is a model made by Mr. Harvey, Department. of Biochemistry, University of Glasgow. A motor drives a rubber belt which rotates the bottles as they rest on small pulley wheels. Each motor drives 40 bottles.

over El6 each. The bottles are filled with 1 1 growth medium and about 10’ cells and placed on a roller machine for cell attachment and growth. The cells can be removed by trypsin treatment but the whole end of the bottle unscrews and the spiral can be removed and the cells scraped off.

44

CELL CULTURE FOR BIOCHEMISTS

Roller bottles are available with corrugated surfaces which increase the growth area several fold. The cells must be removed from such bottles by trypsinisation but may have an advantage for growth of lytic viruses where the infectious process releases the cells from the surface. Abbot Laboratories (Appendix 3) manufacture a Mass Tissue Culture Propagator (MTCP) where the cells grow on glass plates within a large jar through which passes 5% CO, in air (Schleicher, 1973) and Connaught Laboratories (Appendix 3) manufacture a similar Multi-Surface Cell propagator. These pieces of apparatus are expensive and difficult to use but can be made to yield cells in 100 g amounts. However, they can be dismantled which give two major advantages. 1) The cells may be removed from the plates by mechanical means. 2) The apparatus is re-usable and, although initially costly, does not require vast continuous expenditure. However, it puts the onus on the user to prepare the apparatus in a clean and sterile manner. 3.4.2. Cell factories

Nunc (Appendix 3) produces a piece of apparatus rather like ten large trays sealed together - the multi-tray unit or cell factory (Fig. 3.4). The surface area for cell growth is 6000 cm2 and t h s has the advantage that no rolling is required. The cell suspension (about 1.5 1) is introduced by gravity feed from a bottle in less than a minute and the cells (over lo9) can be harvested by trypsinisation. It is important to avoid allowing the medium or trypsin solution to froth, as this causes air locks and spillage, which can easily lead to contamination. It is not possible to remove the cells mechanically unless a way can be developed to dismantle the apparatus using a hot wire. Moreover, the ‘factory’ is very expensive and can be used once only. However, it produces extremely high cell yields using very little space and in contrast to roller cultures requires no machinery which itself is expensive and liable to break down at critical times. As it is

CH. 3.

CULTURE VESSELS

45

Fig. 3.4. A Nunc cell factory. (Courtesy of Nunc and Gibco.)

used only once and is supplied in a sterile pack the chances of contamination are slight. Some laboratories have constructed racks to hold several factories so that one person can seed or harvest them simultaneously. 3.4.3. Perfusion vessels

Various other procedures have been devised to increase the surface area within a vessel upon which cells may grow. The problem is always to maintain an adequate supply of nutrients including oxygen and to remove waste products, particularly acid. The New Brunswick Scientific Co. (Appendix 3) produces a piece of apparatus which continuously perfuses roller bottles by means of a rotating cap through which the various feed tubes pass. A small scale perfusion vessel is available as a sterile pack from Sterilin Ltd. (Appendix 3) for microcinematography. The chamber volume is only 0.4 ml but it may be attached to a heated microscope

46

CELL CULTURE FOR BIOCHEMISTS

stage and individual cells photographed intermittently. Medium may be circulated around a moat surrounding the culture. It is introduced and leaves through needles let into the sides of the chamber (Cruickshank et al., 1959). The coverslip on which the cells grow may be removed and the cells stained or otherwise processed. The ‘Dynacell’ culture system (Millipore cow; Appendix 3) allows medium constituents to diffuse through a 0.6pm membrane into a thin cell growth compartment particuarly suitable for growth of suspension cells. Several units can be coupled together to run from the same perfusing bottle. 3.4.4. Capillary beds

One of the factors limiting the growth of fibroblasts is the availability of medium factors (Dulbecco and Elkington, 1973). Periodic replacement of the nutrient is therefore essential to obtain high cell densities yet has the disadvantage that the composition of the medium bathing the cells is continuously changing. Cell culture on artificial capillary beds does not have this disadvantage but is still not a very popular technique due partly to difficulty in its use and partly to problems with cell harvesting. Different sorts of capillary bed are available made from glass bead columns or hollow fibre systems. Glass bead columns involve circulating medium through a glass column packed with 3-5 mm diameter glass beads. Aeration and monitoring is performed outside the column as with perfusion vessels. The column is first filled with a suspension of cells which, in theory, should attach evenly throughout the column. This, in practice is difficult to achieve. Hollow fibre systems are availabe from Amicon Corp., or BioRad Laboratories based on the semipermeable membranes used in dialysis and concentration cells. Alternatively the Opticel system is based on 1 mm2 pores in a ceramic cartridge. Two sorts of capillaries are used: one for exchange of small molecules in solution and the other of silicone polycarbonate for gaseous exchange. The bundle of capillaries (about 150) is held in a tube into which a cell suspension (about lo6cells in 3 ml medium) is injected by an opening at the side (Fig. 3.5). When the cells have

CH.3

41

CULTURE VESSELS

-

Ports for inoculation and removal of c e l l s

-

Medium equilibrated with 5%C02 in air

art if ic ial capillaries

Medium for recirculation

glass shell

Fig. 3.5. Diagrammatic representation of a capillary perfusion bed.

attached, medium which has been oxygenated and exposed to 5% CO, to adjust the pH, is pumped through the capillary bed at a rate of about 1.0 ml/min. The gases will diffuse through the silicone tubing used to carry the perfusion medium. About 100 ml of perfusion medium is re-used over a period of 2 days before it and the extracapillary medium are changed. The cells remain healthy for a month or more and three small capillary beds in parallel will produce over 2 X 10' cells. This method is extremely useful for the production or metabolic conversion of metabolites. Thus human chorionic gonadotropin may be isolated from the perfusate of human choriocarcinoma cells (Ode11 at al., 1967; Knazek and Gullino, 1973; Knazek et al., 1974).

3.5. Suspension cultures Most cells do not readily grow in suspension, and so this method has limited applicability. However, erythropoietic cells (lymphocytes, hybridomas, and erythroleukaemia cells) show no tendency to adhere to glass or plastic surfaces and will survive if allowed to sit on the bottom of a tube, dish or bottle covered with a shallow layer of medium. Other cells that are grown in suspension have been selected on the basis that they show poor adhesion to the substratum and they continue to grow if maintained in suspension. If not continuously agitated they will, however, settle down and adhere to the substratum. With some cells it is sufficient to maintain them in roller bottles rotating at about 2 r.p.m. but in general special suspension

48

CELL CULTURE FOR BIOCHEMISTS

culture vessels are required and a medium deficient in Mg2+ and Cat+ is used. The suspension culture vessels come in various sizes (from 10 ml to 10 1) and have a magnet suspended just above the bottom of the vessel.

Fig. 3.6. Spinner flasks. The flask on the stirrer is protected from excessive heat by a layer of foam insulation. The vessel on the right is wrapped in aluminium foil for sterilisation. The model with the solid spindle which is allowed to swivel top and bottom is preferable to the one with metal swivel (on the left) as the latter tends to stir erratically.

CH. 3

CULTURE VESSELS

49

Fig. 3.7. Techne spinner flasks and stirrers. T h s type of stirrer produces no heat and can be controlled at low speeds. The suspended ball combined with the raised centre of the flask readily keeps cells or microcarriers in suspension. (Courtesy of Techne.)

The magnet is driven by a magnetic stirrer motor on which the vessel stands. Traditional magnetic stirrers when running for long periods create excessive heat and the vessel requires to be insulated by having a sheet of expanded polystyrene foam interposed between it and the motor. ‘Bellco’ spinner flasks (Fig 3.6) are available from Arnold Horwell Ltd. (Appendix 3). A better system (available from Techne or Wheaton) makes use of a stirrer which generates no heat and will function indefinitely at low speeds. Models are available which will take different numbers of culture vessels whch themselves are of special design (Fig. 3.7). The flask has a central raised region in the base around w h c h moves the magnet encased in a glass ball. This stirring action creates a spiralling movement within the culture medium which keeps all the cells in suspension with a minimum of agitation. The Kontes cytostir vessel is similar. but has blades instead of a ball.

50

CELL CULTURE FOR BIOCHEMISTS

Suspension cultures grow best within a limited concentration range and when the vessel is about half full of culture medium. In order to maintain these conditions it is necessary to give the cultures regular attention. Every day (or in the case of slower growing cells every other day) 50-80% of the suspension should be withdrawn through the side arm and replaced with an equal volume of fresh medium. Stirring vessels for use with suspension cultures have been reviewed by de Bruyne and Morgan (1981) and de Bruyne (1984). Apart from haematopoeitic cells, those cell lines which grow in suspension are exhibiting tumourigenic properties (see 9 2.1). This is often considered undesirable when the cells are being used for production of metabolites (or proteins) to be used for human medication. In fact, their use for such purposes is forbidden by the US Department of Health because of the possibility of introducing potential oncogenic material into human beings (Reaveny, 1985). However, as described in 0 3.2, the traditional vessels used for growing anchorage-dependent cells have limited potential for scaleup which is expensive and cumbersome and is associated with difficulties in sampling and growth control. The realisation of such problems has led to the development of microcarriers.

3.6. Microcarriers Microcarriers are small solid particles (kept in suspension by stirring) upon which cells may grow as a monolayer. They confer the advantage of large scale suspension cultures on anchorage dependent cells. They thus offer the following advantages. 1) High surface to volume ratio which can be readily varied. 2) Large vessels can be used leading to saving in labour costs and space (see 0 3.5). 3) Constant monitoring of both cells and medium is possible. However high levels of lactic acid accumulate at the high cell densities obtained and constant replenishment of the medium is essential together with use of a combination of Hepes and high bicarbonate to maintain the pH.

CH. 3.

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51

4) Harvesting can be easy and does not involve centrifugation (but see 0 4.3.1). 5) Subculture is easy as with suspension cultures (see 0 4.3.1). 6) Microcarriers are very suited to virus production and in some cases virus particles are constantly shed into the medium. 7) Cells may be frozen still attached to the microcarriers. 8) Mitotic cells may be collected by selection of the correct shearing forces (Crespi and Thilly, 1982; Crespi et al., 1981 - see Chapter 11). Microcarriers normally have a density of about 1 so as to facilitate suspension but the all-glass beads available from ICN (Rapid Cell) have a density of about 3 and are not intended for use in suspension cultures but to give an increased surface area when placed in the bottom of a flask (see 0 4.4). The diameter of microcarrier beads is usually about 200pm so the surface area of 1 g of beads is 0.6 m2 which is equivalent to 7 roller bottles, 27 Roux flasks or 315 5-cm Petri dishes (see Table 3.1). With 1 g of beads in 500 ml medium in a suspension flask the surface:volume ratio is 12 compared with 4-6 for traditional vessels which means that medium replacement must take place two to three times as often. Microcarriers fall into three groups: (a) positively charged; (b) negatively charged and (c) non-ionic. 3.6.1. Positively charged microcarriers

Van Wezel (1973) describes the preparation of 100-350pm diameter beads of DEAE-Sephadex A50. Superbeads (Flow Labs) and Cytodex-1 beads (Pharmacia Ltd.; Appendix 3) are based on this formulation. The optimum charge varies with the cell type and if it is too small poor attachment is found. If the charge is too great the strong attachment retards growth. However, a large variety of cell types are reported to grow on Cytodex and Superbeads. The internal charged groups are not essential and tend to bind medium components unnecessarily. The yield of cells can be improved if only the surface layer of the bead is charged for example

52

CELL CULTURE FOR BIOCHEMISTS

with trimethyl-2-hydroxyamminopropylgroups (Cytodex-2, Pharmacia Ltd.) (Gebb et al., 1984). Himes and Hu (1987) investigated the effect of exchange capacity and charge density of microcarriers on cell attachment and spreading. Both attachment and growth were favoured by a higher exchange capacity and attachment occurred more quickly in PBS-A (see Appendix 1) than in growth medium which appeared to compete with the cells for surface binding sites. In fact, 95% of the cells attached to Cytodex-1 beads within 15 min in PBS-A but in medium containing 10% FBS only 22% of the cells attached in the the same time. However, in growth medium, serum is essential for efficient attachment but it can be replaced by factors such as fibronectin, insulin and transferrin (Clark et al., 1980; Clark, 1983) (see 9 5.8.1). Beads may be supplied in suspension in PBS but more usually they are supplied dry and require swelling in PBS-A (100 ml/g) for 3 h and washing in PBS-A and autoclaving (15 min at 15 p.s.i.) before use. In the dry form they are stable for more than 2 years. One gram of Cytodex-1 (dryweight) will swell to about 18 ml containing 7 X lo6 microcarriers with a surface area of 0.6 m2. Bio-Rad Laboratories supply ' Bio-Carriers'. Instead of the crosslinked dextran of the Cytodex microcarriers these have a polyacrylamide matrix with the positively charged dimethylaminopropyl groups linked throughout the matrix. On swelling 1 g dry Bio-Carriers provides 19 ml packed volume containing 7 X lo6 beads with a total surface area of 0.5 m2. Each bead can accommodate 350 cells so that the theoretical value of 2 X l o 9 cells could grow on 1 g Bio-Carriers. For those positively charged microcarriers which swell in aqueous solutions a loading of 3 g dry beads per litre of culture is recommended. 3.6.2. Negatively charged microcarriers

These are made of glass or polystyrene. As glass is much denser than water, the glass beads have only a coating of glass around a polystyrene core.

CH. 3.

53

CULTURE VESSELS

Although cells are negatively charged the electrostatic repulsion is overcome through ionic interactions and protein bridges as with conventional glass and plastic substrata (see 8 2.4). It is thus important for the charge to be on the surface as internal charges can only have a growth retarding effect. Glass-coated and plastic beads are available from Cellon (Bioglas beads), ICN (Rapid Cell G and Rapid Cell P) and Nunc (Biosilon) and do not swell in aqueous solutions. They are sterilised by autoclaving at 121" for 15 min in water.

0

1

2

3

4

5

Dan

Fig. 3.8. L929 cells were seeded in 100 ml GMEM with 10%calf serum at 3 cells per microcarrier bead onto 5 X lo5 C1-(Cytodex 1). P(Biosi1on) or G (Bioglas) beads. The cell number was estimated by releasing the cells with trypsin, briefly allowing the beads to settle and counting the cells with a Coulter counter. Difficulty was encountered releasing the cells from Cytodex, but cells attached only weakly to the glass and particularly to the plastic beads and many cells were free in suspension. The use of electronic cell counters is not recommended for counting cells released from microcarriers as the orifice occasionally becomes blocked by small microcarriers in the suspension.

54

CELL CULTURE FOR BIOCHEMISTS

Because they do not swell 1 g of glass coated or plastic beads will contain only about 230,000 microcarriers of surface area 30 cm2. Bead loadings of 40 g/1 are recommended. 3.6.3. Collagen or gelatin (denatured collagen) beads These may be beads of polystyrene or cross-linked dextran coated with collagen or gelatin (e.g. ICN’s Rapid Cell C and Pharmacia’s Cytodex-3) or the beads may be made completely of cross-linked collagen (ICN’s collagen beads) or gelatin (Ventrex Lab’s Ventregal and KC Biological’s Geli-Bead). The polystyrene beads do not swell in aqueous solutions but the others do. All are sterilised by autoclaving for 15 mm at 15 p.s.i. which will denature the collagen so that when used all will be made of or coated with gelatin. The gelatin surface is a more natural growth surface than glass, plastic or positively charged molecules and this allows seeding at

Fig. 3.9. CHO-KI cells growing on Flow superbeads (Reproduced with kind permission of Dr. David Lewis, Flow Laboratories)

CH. 3.

CULTURE VESSELS

55

lower cell densities (see 0 3.2). Fibronectin binds strongly to gelatin and the gelatin is very sensitive to proteases - particularly collagenase which makes harvesting much easier (see fj 4.5). These microcarriers are particularly suited for growth of epithelial cells. Figure 3.8 shows mouse L929 cells growing on a variety of microcarriers and Fig. 3.9 shows Chinese hamster cells growing on Flow Laboratories 'Superbeads'. Procedures for setting up microcarrier cultures and for subculturing are given in 0 4.4 and 4.5. 3.6.4. Cell entrapment

The advantage of microcarrier culture methods can be obtained for suspension cells by their entrapment within a variety of different beads. Entrapment within beads of calcium alginate is a simple procedure (Familletti and Fredericks, 1988). a) Mix the cell suspension at room temperature with 2 volumes of 1.4%sodium alginate in 0.1M NaC1. b) Using a 21 gauge needle add the suspension dropwise to a solution of CaC1, (50 mM) and NaCl (0.1 M) at pH 7.2. Beads (2-3 mm diameter) are formed as the calcium alginate precipitates. c) Wash the beads three times with growth medium. Larger beads (e.g. 8 mm diameter) can be made by increasing the bore of the needle, and inclusion of gelatin (5%) in the cell suspension leads to beads containing hollow cavities as the gelatin dissolves during culture. Agarose beads are also popular and the method of entrapment has been described by Nilsson et al. (1983).

3.7. Air lift systems Cells growing in suspension or on microcarriers can be maintained in suspension by introducing a gas at the bottom of the culture. As

56

CELL CULTURE FOR BIOCHEMISTS

the bubbles rise they cause circulation of the medium and uniform distribution of the cells. Ventrex (Appendix 3) market a disposable cell lift system whch takes up to 650 ml suspension. It can be used in batch or continuous feed and a heating jacket is available which means it does not need to be operated in a hot room. Kontes (Appendix 3) sell a similar, reusable ‘Cytolift’ bioreactor. The advantages of an air lift system are that cells are not damaged by contact with stirring mechanism and that very efficient gaseous exchange is possible. The disadvantage is that foaming can be a problem and special anti-foam agents are required, e.g. MS antifoam emulsion RD (Hopkins and Williams, Appendix 3) or various antifoams sold by Kontes (Appendix 3).

3.8. Bioreactors Scale-up of cell cultures makes use of suspension cultures (erythropoietic cells or microcarriers) or, less often use of capillary beds (hollow fibre systems or glass bead columns), but these suffer from the same disadvantages seen with smaller scale cultures (0 3.4.4). In particular, nutrients are depleted as the medium flows through long columns or beds and high rates of flow coupled with recirculation are often employed. Nevertheless, Organon have used a hollow fibre dialysis system for production of monoclonal antibodies (Schonherr et al., 1985). Invitron’s hollow fibre system has been used to produce cell conditioned media and the Cell-Pharm System (Jencons Ltd.; Appendix 3) will produce up to 20 g cell secreted product per month. Techne sell a bioreactor for growing cells in suspension in volumes up to 3 1 (Fig. 3.10) and New Brunswick has a model which will take 5 1. The Techne bioreactor maintains the cells or microcarriers in suspension with a floating stirrer but such a mechanism becomes increasingly less efficient up to 20 1. pH, PO, and temperature are monitored and controlled and the reactor can be used for batch cultures or in continuous mode.

CH. 3.

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57

Fig. 3.10. A bioreactor can be used for growth and monitoring of 3 1 of cells.in the laboratory and industrial models take up to 1000 1. The figure shows the Techne bioreactor. (Courtesy of Techne.)

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CELL CULTURE FOR BIOCHEMISTS

The CelliGen (New Brunswick) has four possible modes of gas delivery coupled with suspension by air lift or a variety of impellers. Large, slowly rotating impellers are preferable to avoid damage to cells. Densities up to 5 X lo7cells per ml can be obtained (Vosper, 1987). Bellco (Appendix 3) produce a 3 1 air lift bioreactor designed initially for use with hybridoma cells entrapped within alginate macro beads (6 3.6.4). In this system the rate of antibody production was over 10 times that achieved in a spinner culture (Familletti, 1987). A stainless steel matrix can be fitted into Bellco’s bioreactor and this provides an ideal substrate for anchorage dependent cells. This turns the bioreactor into a capillary diffusion system. Butler (1987) has considered the physics of gaseous exchange and rate of nutrient supply and concludes that a 100 1 culture running in the continuous mode may be more efficient than a 1000 1 batch system. Different systems for large scale production have been compared by van Wezel(1985)who concludes that continuous perfusion systems are the most efficient for production of cellular components.

CHAPTER 4

Subculturing 4. I . Dissociation techniques Cells in tissues and cells growing as monolayers on glass or plastic surfaces are held together and to the substratum by mucoproteins, and sometimes by collagen (see 0 2.4). In addition, many cell monolayers and tissues, especially epithelial tissues, require divalent cations (Ca2+,Mg2+)for their integrity. Thus for tissue dissociation or for releasing cells from monolayers various protease solutions are used, sometimes in association with chelating agents. 4.1. I. Ttypsin

For many years a solution of crude acetone powder of bovine or porcine pancreas has been used to disaggregate tissues and to release cultured cells from their substratum. These preparations - referred to as trypsin 1: 250 (based on an international standard) - contain not only trypsin but a range of enzymes including chymotrypsin and elastase which are equally important. Purified trypsin seldom is as efficient as the cruder preparations, especially on tissue disaggregation, when mucinous clumps result (Ronaldoni, 1959). Prolonged exposure to trypsin should be avoided as this damages the cells. The best way to inactive the trypsin when the desired dissociation has been achieved is to add serum (or medium containing serum) which contains a natural trypsin inhibitor. Trypsin solutions are usually made up in saline solution (PBS-A) (see Appendix 1)or in saline-citrate and are used as 0.25% solutions. To make 1 1 trypsin-citrate dissolve trypsin (Difco 1:250) as follows 59

60

CELL CULTURE FOR BIOCHEMISTS

by vigorous stirring for 3-4 h: trypsin (1:250) tri-sodium citrate sodium chloride phenol red (1%) distilled water

2.5 g 2.96 g 6.15 g 1.5 ml to 1 1

Adjust the pH to 7.8 with NaOH (approx. 2 ml 1 N NaOH) and filter several times through Whatman’s No.1 filter paper. Sterilise by filtration using a prefilter and an 0.22 pm pore size membrane. This stock solution is conveniently stored frozen at - 20°C in 20 ml and 80 ml amounts after checking for contamination using 1) Saboraud fluid medium at 31°C for 1 week (Appendix 4); 2) brain heart infusion broth at 37°C for 1 week (Appendix 4). (One check of each should contain calf serum). 4.1.2. Pronase Pronase is recommended for primary cultures as it gives a better single cell suspension than trypsin (Gwatkm, 1973). It is not always as good with cell lines. An 0.25% solution is made by dissolving 1.25 g standard B grade pronase (Calbiochem Corp.) in 500 ml PBS-A at room temperature. After 30 min the cloudy solution is centrifuged (100 g, 10 min) and sterilised by filtration as for trypsin. 4. I . 3. Collagenase

Collagenase is reported to cause least damage and is used in the preparation of clonal cell cultures (Hilfer, 1973). Combined use of collagenase and hyaluronidase followed by trypsin or pronase without vigorous agitation (which leads to loss of cell viability) was satisfactory for mouse mammary gland cells (Prop and Wiepjes, 1973). Preparations of collagenase vary. Worthington (Appendix 3) supply four varieties of crude collagenase, each of which is suited to isolation of cells from a particular tissue. Individual batches of enzyme can be reserved and tested.

CH. 4.

61

SUBCULTURING

4.1.4. Dispase

Dispase is a neutral protease from Bacillus pofymyxa available from Boehringer Corp. Ltd. It is particularly suitable for disaggregation of animal tissues, which seldom suffer from prolonged treatment. It is available alone, or mixed with collagenase. It requires Ca2+ for activity and its action is readily neutralised by EDTA. 4.1.5. EDTA (Versene)

Treatment with chelating agents such as EDTA which removes divalent ions, leads to dissociation of cell monolayers and release of the cells into suspension without protease action. Perfusion of rat liver with citrate solutions has also been used in attempts to produce primary liver cell cultures. Very often, however, mixtures of trypsin or trypsin-citrate with versene are used to release cells from monolayer cultures as the combination works very much faster than either alone. In our laboratory the following procedure is used to make up 10 1 of versene solution in PBS-A: NaCl KC1 Na HPO, KH PO, Versene (diaminoethanetetraacetic acid disodium slat) Phenol Red 1% Distilled water to

10 litres 80 g 2.0 g 11.5 g 2.0 g

2.0 g 15 ml 10 1

Dispense in 20 ml, 80 ml or 260 ml amounts. Autoclave at 15 lb pressure for 15 min for 20 ml bottles. Autoclave at 15 lb pressure for 20 min for 80 ml and 160 ml bottles. Store at room temperature.

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CELL CULTURE FOR BIOCHEMISTS

To make trypsin-versene mix: 1 volume 0.25% trypsin or trypsin-citrate (see above) 4 volumes versene 4.1.6. Mechanical means

For many biochemical studies it is undesirable to release the cells from the substratum using trypsin. This is especially true in two circumstances. 1) Studies on cellular surfaces. Trypsin leads to loss of surface proteins including glycoproteins and other antigens and it is these changes which presumably lead to cell death when trypsinisation is prolonged. 2) Studies involving timed incubations with drugs, radioactive precursors, hormones, etc. In these instances it is important to harvest the cells promptly without exposure at 37OC to an altered environment for an indefinite period. For these studies cells must be harvested mechanically, e.g. by scraping with a rubber policeman or by ,the use of chemicals (e.g. alkali, acid or detergent) which lead to instantaneous death of the cells from which various products may then be isolated.

Fig. 4.1. Scrapers used to remove cells from surfaces. The upper scraper is used to scrape cells from inside roller bottles. The blade folds forwards allowing it to pass through the narrow neck. By pushing against the bottom of the bottle the blade opens as shown and the shape of the arm prevents further movement. The middle scraper is a rubber policeman fitted to a bent glass rod. It is used for scraping cells from smaller bottles. The lower scraper is used for dishes where the edges of the cut silicone bung allow cells to be removed efficiently from the corners of the dish.

CH. 4.

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63

Rubber policemen are simply rubber sleeves (obtained from MacFarlane Robson Ltd; Appendix 3), which fit over the ends of glass rods and provide a soft surface with which cells may be scraped from their substratum. Alternatives for use with dishes are wedges of silicone rubber cut from bungs and stuck on hypodermic needles (Fig. 4.1). These also have the advantage that they can be readily sterilised by autoclaving. A collapsible type of windscreen wiper (Fig. 4.1) is readily constructed for scraping cells from the inside of roller bottles. Scrapers can be obtained from Costar (Appendix 3).

4.2. Subculture of a cell monolayer a) Prepare a bottle of suitable growth medium and warm to 37°C. b) To 20 ml versene add 5 ml trypsin-citrate solution and warm to 37OC. c) Examine the monolayer macroscopically and microscopically for any signs of bacterial contamination (Chapter 9). d) Aseptically pour off the medium into a universal bottle. This is for a bacteria check (see below). e) Add a small volume (1 ml for a 25 cm2 flask; 5 ml for a 75 cm2 flask) of trypsin-versene solution at 37°C. f ) Immediately remove this trypsin-versene and add a further small volume of fresh trypsin-versene at 37OC. g) Replace the cap and incubate the bottle at 37°C. After a few minutes the cells become released into suspension. Thts process may be aided by gently tapping the base of the bottle with the palm of the hand. h) DO NOT LEAVE THE CELLS IN TRYPSIN FOR LONGER THAN NECESSARY AS IT WILL DAMAGE THE CELLS. i) To prevent excessive trypsin astion add 5-20 ml growth medium containing serum. j) Pipette gently to complete dispersal and count the cells (see below and 0 7.2). k) Transfer the appropriate volume of cell suspension (see Table 3.1) into new bottles and dishes and add fresh medium. Usually cells

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CELL CULTURE FOR BIOCHEMISTS

may be split 1: 4 every 2-3 days but some strains grow better if split 1:2 and some cell lines may be split 1: 10. 1) Gas the bottles with 5% CO, in air (see Q 3.1.2) and incubate at 37°C. 4.2.1. Viable cell count

Take some cell suspension and dilute with an equal volume of 0.1% trypan blue (stains dead cells: Appendix 2). Take up some trypan blue cell suspension in a Pasteur pipette and fill a haemocytometer counting chamber by capillary attraction (6 7.2.1). Take care not to flood the channels of the chamber. Count the cells under X 10 objective. Count the four sets of 16 small squares (on each corner of the central ruled area (8 7.2.1). Count only unstained cells. Divide by 4 to give the average count per 1 mm2. Since the area is 1 mm2 and the depth 0.1 mm, the conversion factor for the counting chamber is lo4, e.g. let the average count = 20 cells per 1 mm2 20 X lo4 = number of cells per ml of trypan blue suspension 2 x 20 x l o 4 = number of cells per ml of original suspension i.e. 4 x 105cells per d. 4.2.2. Bacterial check

Take the medium from step d of the subculture procedure and centrifuge at 800 g for 15 min. Discard the supernatant. Using a platinum loop, place the sediment on a blood agar plate (Appendix 4) and incubate at 37°C for at least 2 days to check for bacterial growth (Chapter 9).

4.3. Subculture of cells growing in suspension Cells growing in true suspension (i.e. not attached to microcarriers) are simply subcultured by dilution. a) Prepare a bottle of suitable growth medium and warm to 37°C. b) Remove 75-90% of the cell suspension. This may be processed to

CH. 4.

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65

obtain either the cells or the culture supernatant by centrifugation at 1000 r.p.m. for 5 min. c) Replace with fresh medium to restore the original volume. Alternatively, the volume of cell suspension may be increased by adding fresh medium and increasing the size of the culture vessel as and when necessary.

4.4. Protocol for setting up microcarrier cultures (see 5 3.6) Pharmacia produce a booklet with many useful hints on microcarrier cell culture. 4.4.1, Preparation of the microcarriers

1) Swell the dry beads made of cross-linked dextran, polyacrylamide or gelatin in PBS-A or Hepes buffered saline as described by the manufacturer. The cross-linked dextran beads require 2-3 h to swell at room temperature. Avoid stimng with a simple bar magnet as this may grind the beads. Addition of non-ionic detergent (e.g. Tween 80) to 0.1% may help initial wetting of the microcarrier. 2) Wash the beads by decantation and resuspend in the swelling buffer. Glass and plastic beads can be suspended in water. 3) Dispense into appropriate bottles at about 1 g per bottle for the beads that swell or 10 g per bottle for the glass and plastic beads. 4) Autoclave at 15 p.s.i. for 15-20 min and store at 4°C. 5 ) Before use, decant the supernatant and soak the beads in 2-3 volumes of warm culture medium for 30 min. 4.4.2. Preparation of the culture vessel

All glass and plastic-ware must be siliconised before use to prevent adherance of cells and microcarriers to the culture vessel. 1) Coat the clean, dry glassware with Repelcote (Hopkins and Williams Ltd.), dimethylchlorosilane (British Drugs Houses) or similar reagent.

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CELL CULTURE FOR BIOCHEMISTS

2) Dry at 37°C to remove solvent. 3) Wash twice with distilled water and sterilise by autoclaving (Chapter 8). It is helpful to place a sheet of aluminium foil under the caps of the culture vessel as this prevents the liners sticking to the glass necks. 4.4.3, Initiating a culture

The number of cells per microcarrier bead, the concentration of beads and the initial stirring conditions can vary dramatically. The following is a suggested protocol but if plating efficiency is poor the culture volume and speed of stirring can be decreased and the number of cells per microcarrier increased. 1) Transfer the microcarriers to the culture vessel in the starting volume of growth medium, gas with 5% CO, in air (see 9 3.1.2) and equilibrate at 37°C. A suitable volume of medium for a 1 1 flask is 50 ml containing about lo7 microcarriers. 2) Add the cell suspension in an equal volume (i.e. 50 ml) of growth medium. A reasonable starting value is three cells per bead and so around 3 x lo7cells should be added. 3) Stir the culture intermittently at 30 r.p.m.. A suitable intermittent procedure is to stir for 1 min every 30 min. 4) The cells should all attach within 4-6 h after which a further 150 ml prewarmed and pregassed growth medium should be added and the culture stirred continuously at 60 r.p.m.. This speed may need to be increased to 80 r.p.m. for the larger or denser microcarriers but should be the minimum required to maintain the beads in suspension. 5 ) After 1 or 2 days add a further 250 ml prewarmed growth medium and continue stirring at 60 r.p.m.. This is the final volume, and the culture can be maintained at this volume by subculturing as described in 9 4.5. 4.4.4. Non-stirred microcarriers

A layer of microcarriers can be arranged on the growth surface of a flask or roller bottle to increase the surface area. In this case the

CH. 4.

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61

flask or bottle must not be siliconised. Two alternative procedures can be used: 1) to a newly seeded flask add sufficient microcarriers to almost cover the bottom of the flask, or 2) to a flask in which the cells are 50% confluent add microcarriers to cover about half the area of the flask. If the culture vessel is a roller bottle, rotation should be very slow (0.1 r.p.m.) until the cells and beads are attached.

4.5. Subculture of cells growing on microcarriers (see 9 3.6) It is often difficult to remove cells from dextran microcarriers by trypsinisation and for this reason two new types of bead have been introduced. The glass or plastic bead can be trypsinised in a manner similar to a glass bottle while the ‘Gelibead’ is itself digested by trypsin and collagenase leaving behind a suspension of cells. The glass beads can be reused by soalung in 1 M NaOH for 30 min, rinsing very well in distilled water and autoclaving in water. The following protocol can be used to isolate cells from polydextran (Cytodex-1) or polyacrylamide (Biosi1on)microcarriers. a) Allow the beads to settle and remove the culture medium. b) Rinse the beads in 0.25% trypsin in PBS-A or in trypsin/versene (0 4.1.5). c) Stir in trypsin/versene (30 ml/g microcarrier) at 37°C while monitoring with the microscope for release of cells. This may require further vigorous pipetting especially for epithelial cells which typically require more than 10 min for complete release. d) Add serum-containing growth medium to neutralise the trypsin. e) Allow the beads to settle and remove the cell suspension. The separation of cells from beads can also be accomplished by passing the suspension through a seive with a 75 or 90 pm mesh depending on bead diameter. f’) Seed onto new microcarriers (5 4.4.3).

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For gelatin (Cytodex-3) or collagen (Rapid Cell-C) coated beads collagenase can replace the trypsin/versene and either can be used to release cells from glass (Bioglas, Rapid Cell-G) or plastic (Bio-carriers, Rapid Cell-P) microcarriers. When using collagenase the beads should first be rinsed in EDTA in PBS-A (0 4.1.5) and then stirred at 37OC in a solution of 0.01-0.1% collagenase in PBS containing calcium and magnesium (Appendix 1, Table 2) for about 15 min. Gelibeads (KC Biological and Sterilin), Cellagen (ICN Biomedicals Ltd.) and Ventregel (Ventrex Labs Inc. and Tissue Culture Services) are 100% cross-linked gelatin and are completely digested by collagenase. This aids liberation of the cells and obviates the need to separate the beads from the cells as the former disintegrate completely. Microcarriers have an advantage over flasks and dishes in that it is not essential to liberate the cells from their substratum in order to subculture them. Whether the beads are kept in suspension or allowed to settle on the surface of a flask or bottle they can be subcultured as follows: a) If not already suspended put the beads into suspension by agitating the flask (this may require vigorous shaking if the beads are firmly attached to the flask with the growing cells - 0 4.4.4). b) Remove a sample of the bead suspension to a fresh flask containing growth medium and some new beads (the amount of new beads will vary with requirements, but for a non-stirred culture they should cover about one quarter of the surface area of the flask). c) Allow the beads to remain on the bottom of the flask at 37"C, or stir intermittently for up to 24 h (see 0 4.4.3). Then add more medium and stir continuously. d) Further additions of fresh beads may be made as required. On each addition a period of intermittent stirring should occur to allow transfer of cells onto the new beads.

4.6. The growth cycle On subculturing cells into new vessels they do not start to grow immediately at their maximum rate especially if they are seeded as a

CH. 4.

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69

dilute inoculum. Rather, they exhibit a lag phase of 1-2 days while they adapt to their new environment and condition the new medium. That this is not an intrinsic characteristic of cells was shown by Puck (1972). The lag phase can be almost entirely eliminated by taking care not to over-trypsininse and to maintain the cells at 37"C, and with mouse L929 cells most cells will divide within a day when more concentrated inocula are used. However, it is often found that little increase in cell number occurs on the first day following subculture; only after 2 days have the cells doubled in number. Thereafter, exponential growth may be maintained for a further 2-10 days if there is sufficient room in the vessel and the medium is changed regularly. Before long, however, the rate of growth falls and the cell number plateaus (Fig. 4.2). Some cells remain viable in this state for up to several weeks, especially if the medium is changed every few days, but with other cell lines growing in so-called monolayer culture, the cells pile up one on top of the other, the lower cells become starved and soon the cell sheet peels off the glass. Changing the medium in stationary monolayer cultures prevents the build-up of acid and other waste products and supplies fresh nutrients. In cells which have just entered the plateau it also has the effect of stimulating some of the cells to undergo a new round of DNA synthesis and cell division. This is particularly marked in mouse L929 cells as regular feeding enables a very h g h density monolayer to form in which all the cells are of a small uniform size. With monolayer cultures it is uncommon to extend the length of the experimental phase of growth for more than a few days. Seeding the cells at lower densities usually leads to a longer lag phase during which some cell division occurs but the generation time is extended and, in general, an 8-fold increase in cell numbers (i.e. 3 cell divisions) is all that is achieved during the exponential phase. However, as stated above, Puck (1972) has achieved reproducible exponential growth from single HeLa cells which continued for more than 10 days and gave rise to a 2000-fold increase in cell number. Some cell types specify, in their name, the procedure required at subculture. Thus 3T3 cells should be split 1 to 3 every 3 days to maintain them in optimal conditions and prevent their ever forming a stationary, confluent culture.

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5.0r

. E a

U

In

aJ 5

c C

r

0

I 1

I

I

3

5

7

9

I

11

Time (days)

Fig. 4.2. Growth cycle of mouse L929 cells. Mouse L929 cells were inoculated into 2 oz medical flat bottles (5x10’ cells in 5 ml Eagle’s Minimal Essential Medium containing 10%calf serum). The medium was changed every two days. Bottles were incubated for 60 min with 2 pCi (6-3H)-thymidine (80 pCi/pmol) at a final concentration of 5 pM after which they were harvested by trypsinisation, their A) estimated using a Coulter counter and the rate of DNA synthesis number (A(00 ) estimated from the incorporation of [’HI thymidine (Chapter 12). Courtesy of Dr. J.G. Lindsay, 1969.

In suspension, cells may be maintained in exponential growth for long periods, and in theory indefinititely if the cells are grown in a chemostat where the small trickle of incoming nutrients is balanced by the outflow of cell suspension thus maintaining a constant environment and constant cell number. More usually a certain volume of cell suspension will be removed every day (or every other day) and replaced with fresh medium. However, in long-term cultures, contamination is a serious hazard and cells in suspension are usually grown in batch cultures which exhibit similar growth kinetics to monolayer cultures.

CHAPTER 5

Cell culture media 5.I. Introduction It is no longer common for laboratories to make up media from their individual constituents. Even when a special medium devoid of one particular amino acid is required it can usually be found in a commercial suppliers catalogue. The advantage of using commercially available media far outweigh their increased cost to all except those involved in studying the media themselves. For this reason detailed methods of media preparation from individual components are not given here. If required these may be found in Morton (1970) or in the original references. It cannot be too strongly stressed, however, that when preparing media, water must first be glass distilled and then deionised (always check the cartridge) and stored in a glass or plastic container. The Milli-Q system (Millipore Ltd., Appendix 3 ) provides water of consistently high quality. In the early work, embryo and tissue extracts were commonly used to encourage growth of cells but now these are only occasionally added to medium and then only for cultivation of certain primary cells (e.g. see 0 2.2 and 0 2.3). Today’s media, however, as well as containing amino acids, vitamins, salts and glucose normally include serum at about 10%and sometimes also other additives such as Bactopeptone or tryptose phosphate (Appendix 1). Starting with medium 199 (Morgan et al., 1950) which contains over 60 synthetic ingredients (Appendix 1: Table 18), many media formulations are available commercially. Unfortunately, the details of the methods of preparation and the absolute amounts of the various ingredients tend to vary from supplier to supplier. Morton (1970) has surveyed a number of commercially available media listing the initial formulation and preparation method. In 1955, Eagle identified the growth requirements for several mammalian cell lines in both qualitative and quantitative terms. His 71

12

CELLCULTURE FOR BIOCHEMISTS

basal medium (BME - Appendix 1: Table 12) has 28 ingredients and is supplemented with 5% bovine serum. He later modified this medium to enable cells to grow without daily treatment and his minimum essential medium (MEM) is widely used today (Eagle, 1959). Further modifications have been suggested by other workers, e.g. Glasgow MEM, which is essentially twice the basal medium (BME) concentration of amino acids and vitamins with extra glucose and bicarbonate, has been recommended for the growth of BHK21/C13 hamster cells (McPherson and Stoker, 1962), and Dulbecco’s modification (DMEM), which contains four times the BME level of amino acids and vitamins together with some non-essential amino acids and added ferric nitrate, is recommended for propagating polyoma virus in hamster cells (Dulbecco and Freeman, 1969) (see Appendix 1: Table 12). A series of new media have been introduced in an attempt to grow cells in a completely defined or serum-free environment and these are considered in 0 5.8. When deciding which medium to use for a particular cell one must distinguish whether or not yield of cells or yield of some cell product is the more important. Often the two requirements are antagonistic such as when differentiated-cell products are being studied. In such a case medium selection must be linked to assay of the desired product. When high cell number or rapid growth is desired this can be measured directly (6 7.2) or by assay of the rate of DNA synthess (0 12.1) or accumulation of cellular material. There are a number of methods for estimating the total amount of protein, or nucleic acid in a cell culture and these are considered in Appendix 5, but the method of using methylene blue to assay for total cellular nucleic acid has been adapted for use in 96-well plates and allows simultaneous rapid screening of many different media using microtitre equipment (Pelletier et al., 1988).

5.2. Balanced salt solutions Media are based on a balanced salt solution (BSS) which, as well as supplying essential ions, is also important to maintain the osmotic

CH. 5 .

CELL CULTURE MEDIA

13

balance. In addition, glucose is sometimes included in balanced salt solutions. However, it is better to add a sterile glucose solution along with filter sterilised sodium bicarbonate after the other ingredients have been sterilised by autoclaving. Salt solutions have developed over the years, but Earle’s BSS and Hanks’ BSS together with one devised by Eagle for suspension cultures are those in common use today. These are usually made up as 10-fold (10 x ) concentrates without bicarbonate in glass distilled water and they can be stored in the cold room with a drop of chloroform in the bottom of the bottle to prevent bacterial growth (Appendix 1: Table 1). The stock solution is diluted with distilled water and autoclaved, after which bicarbonate and glucose may be added. The details of how to make up Earle’s and Hanks’ BSS are in Appendix 1 (Table 1). The glucose and bicarbonate solutions are sterilised separately by filtration, and details of their preparation are given in Appendix 1 (Tables 3 and 4). It is very important to maintain the correct pH of growth media between 7.3 and 7.5 and this is generally achieved with a bicarbonate/CO, system. In the presence of 5% CO, in air this produces a pH of 7.4 (the phenol red imparts a tomato colour to the medium at this pH; if the medium becomes too alkaline it turns red and then puce while when it is too acid it becomes yellow). As growing cells produce acid it is sometimes necessary to increase the level of bicarbonate in the medium in an attempt to maintain pH. The phosphate in BSS also helps to maintain the pH as well as supply essential phosphate ions. As well as being essential for certain reactions N a + and, to a lesser extent, K + are important in maintaining the osmotic balance in the cell. The sodium ion concentration is maintained about isotonic largely by the NaCl in the BSS but in Earle’s BSS this is supplemented with NaHCO, and to a lesser extent with other salts (see Appendix 1). If the N a + concentration is increased from the normal 120 mM up to 220 mM it leads to a dissociation of polysomes with concomitant inhibition of protein synthesis and cell growth (Fig. 5.1) (Saborio et al., 1974). Calcium ions are essential for the attachment of cells to a glass or plastic surface and hence are omitted from BSS for use in suspension

14

CELL CULTURE FOR BIOCHEMISTS

Time ( h )

Time ( h )

Fig. 5.1. Effect of increased NaCl concentration of the incorporation of ['Hluridine and [3H]leucine into CHO cells. Hamster CHO cells were set up as coverslip cultures in a multiwell dish in Eagle's medium (Glasgow modification) containing 10% calf serum (lo5 cells/0.5 ml). After overnight growth, 10 p1 of [3H]uridine (0.5 pCi) or [3H]leucine (2 pCi) was added per well, and 30 min later 10 p l 5 M NaCl was added to half the wells. Coverslips were harvested at the indicated times and washed twice in ice-cold BSS, 4 times in cold 5% TCA and twice in absolute ethanol. The cells were dissolved in 0.5 ml hyamine hydroxide and the radioactivity counted using a scintillaControl, i.e. 120 m M NaCI; 0 , 220 mM tor of 0.5% diphenyloxazole in toluene. NaCI.

.,

cultures (9 3.5) and usually the phosphate concentration is raised 10-fold (Eagle, 1959). Dulbecco's phosphate buffered saline (PBS) is similar to Hanks' BSS but bicarbonate is omitted and the levesl of Na,HPO, and KH,PO, are raised to provide increased buffering capacity. It is not used as a basis for growth medium but is often used for washing cell monolayers. PBS is made up in three parts (Appendix 1: Table 2). PBS solution A (PBS-A) lacks the Ca2+ and Mg2+ ions which are

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CELL CULTURE MEDIA

added separately as PBS solution B and PBS solution C, respectively, to constitute PBS proper. 5.2.1. Zwitterionic buffers

Hepes has been shown to be non-toxic to cells and can be used instead of bicarbonate in which case cells need not be maintained in an atmosphere of 5% CO, in air. However, as bicarbonate is essential for cloning cells (see 0 7.1) mixtures of Hepes and bicarbonate are used and the cells grown in an atmosphere of 2% CO, in air. Hepes is 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid. HO.CH,.CH,-+NH

n

N-CH,-CH,SO;

u

MW=238.3

At 37°C it has pK,, = 3 and pK,, = 7.31. It is thus a much more suitable buffer than bicarbonate which has a pK = 6.10. Hepes is used at 10-25 mM and is added to medium from a stock solution (1 M) whose preparation is described in Appendix 1, Table A1.5. As well as Hepes, other zwitterionic buffers have been used in cell culture medium. TRICINE ( N-[Tris-hydroxymethy1)-methyllglycine, pK, = 7.79 at 37°C) has been used in Eagle’s MEM (Spendlove et al., 1971) and in Swim’s 577 (Gardner, 1969) and TES, (N-[(Trishydroxymethyl)methyl]-2-aminoethanesulphonic acid, pK a = 7.16 at 37°C) has been used in Eagle’s MEM, BME and in medium 199 (Williamson and Cox, 1968; Massie et al., 1972). These buffers are used in varying concentrations (10-50 mM). Eagle (1971) suggests combinations of buffers which can be used to buffer the medium over the range 6.4-8.35.

5.3. Eagle’s medium This, in its various forms (e.g. BME, MEM; see Appendix 1; Table 12), is generally suitable for the cultivation of most cell lines for

16

CELL CULTURE FOR BIOCHEMISTS

which it is supplemented with 10% calf or foetal calf serum and occasionally with tryptose phosphate. Basal medium (BME) needs to be changed at least every other day to support continued cell growth while MEM supports growth for several days. Eagle’s media contain, in addition to the salts and glucose of BSS, 12 essential amino acids and 9 vitamins. Glutamine and, if required, antibiotics along with serum etc., have to be added also before use. It is common practice when culturing certain cells, e.g. BSCl and CV1 monkey cells, to supplement Eagles MEM with certain non-essential amino acids (Appendix 1). The different forms of Eagle’s medium are available commercially; for example from Flow Laboratories or Gibco (Appendix 3). Complete 1 X MEM medium only requires the addition of glutamine, antibiotics and serum, and this is very suitable for small scale work in laboratories which lack facilities for preparing media. It is, however, a very expensive way of buying media if more than a litre or so is required per month. 10 x MEM medium simply requires dilution into sterile distilled water followed by addition of glutamine, serum, bicarbonate and antibiotics, if required. 50 and 1 O O x concentrates of vitamins and amino acids may either be added to the appropriate volume of sterile BSS and glutamine and serum added, or they may be mixed to make a 10 x MEM stock (Appendix 1: Tables 13 and 14). This is a suitable way of making medium as the glucose and glutamine may be incorporated into the 10 X MEM stock. It is significantly cheaper than buying Eagle’s 10 x MEM, yet occupies much less cold room space than if 1 X medium were reconstituted initially. The methods of preparation of 10 x stocks of Eagle’s MEM and Dulbecco’s MEM are indicated in Appendix 1 (Tables 13 and 14). These methods involve filtration of the 10 X MEM, but this can be avoided if the glucose and glutamine are dissolved separately and sterilised by filtration and the reconstitution is carried out aseptically using sterile distilled water. Both of these approaches do introduce possible entry points for contamination, and only the large users are recommended to prepare their own media.

C H . 5.

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I7

It should be remembered that glutamine is unstable and stock solutions have a limited shelf life. Growth media, i.e. media suitable for the continued culture of established cell lines, are prepared by dilution of the l o x stock solutions. If the 10 X stock is made with salts present (i.e. if it is obtained commercially or if it is made from powder - see below) then it must be diluted with water. If, however, it is made as described in Appendix 1, Table 13 or 14, it must be diluted with BSS as indicated in Table 15. The latter table gives methods of preparing various ‘minimal essential media’ supplemented with calf or foetal calf serum and in one case also with ‘non-essential amino acids’. The various media are all based on Eagle’s MEM and have been developed to improve the growth of certain cell lines or strains as indicated. 53.1. Powdered media

The simpler forms of Eagle’s media (and some other media) are available commercially in powdered form from for example Flow Laboratories or Gibco (see Appendix 3). This is the cheapest way of buying media if large quantities are used; the powder occupies little space and may be stored in the cold room in sealed containers for 6-12 months. Powdered medium should be reconstituted at room temperature as per maufacturers instructions, which leads to 1 X medium. It is important not to leave traces of powder undissolved. Bicarbonate is added separately and the pH adjusted to about 7.1 before sterilisation by filtration. Before use, glutamine should be added, if not already present, together with serum (usually to lo%, as described in Appendix 1). Each batch should be checked for bacterial contamination in a) Sabaraud fluid medium at 31°C for 1 week (Appendix 4); and b) brain heart infusion broth at 37°C for 1 week (Appendix 4). Although not generally recommended by the maufacturer, if care is taken to ensure that all the powder is dissolved we have found that Glasgow MEM powder can be reconstituted to give a 10 X stock

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which then only requires dilution with sterile distilled water and p H adjustment before use. In this case it is important not to add bicarbonate or adjust the pH until the medium has been diluted. The advantages of reconstituting to 10 X are: 1) there is a smaller volume to filter sterilise; 2) the 10 X stock occupies less cold room space; 3) it is more economical to buy bottles which reconstitute to 5 1 of 10 x stock (50 1 of 1 x medium) than smaller amounts. The requirement to filter large volumes of media is circumvented by use of autoclavable powdered medium Here the phosphate buffer has been replaced with a succinate buffer (Yamane et al., 1968). The powder should be dissolved in 95% of the final volume of distilled water and the pH adjusted to 4.1 before autoclaving for 15 min at 121°C. After cooling to room temperature 3 ml sterile 7.5% NaHCO, is added per 95 ml medium, together with glutamine and serum etc., and the pH is adjusted to 7.2-7.4 with sterile 1 N NaOH, if necessary. The problems associated with preparing one’s own media have led to all but the largest users buying X l or X 1 0 medium from commercial suppliers. This removes the need for exhaustive checking with the discovery of occasional unsatisfactory batches and consequent financial loss.

5.4. More complicated media Different media have been developed for specific cell lines in order to obtain optimal growth or in an attempt to grow cells in defined media without the addition of serum. Thus McCoy’s medium 5A (McCoy et al., 1959) has been used as a standard medium for cloning cells. It is based on BME amino acids and the vitamins from medium 199 (Appendix 1: Table 18). This was modified further to form RPMI (Rosswell Park Memorial Institute)-1629 (Appendix 1: Table 17) for long-term culture of leukaemic myoblasts (Armstrong, 1966). Ham’s F10 medium was very carefully formulated for cloning diploid hamster ovary cells (Ham, 1963) and was later modified Ham’s F12 (Appendix 1: Table 16) (Ham, 1965) - to support

CH. 5 .

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19

growth with addition of minimal amounts of serum. This has been further modified for growth of 3T3 cells in the absence of serum (MCDB402; Shipley and Ham, 1981). The Connaught Medical Research Laboratories Media are also designed to support cell growth without the addition of serum. Thus CMRL 1066 (Appendix 1: Table 20) will support the growth of several cell types as will the National Cancer Institute medium NCTC 135 (Appendix 1: Table 19) and CMRL 1415 comes nearest to supporting the growth of cell strains (Healy and Parker, 1966a,b; Evans et al., 1964). See 8 5.8 for more details. Many of these more complex media are available from commercial suppliers, e.g. Flow Laboratories or Gibco (see Appendix 3) at 1 x formulation, and the constitutions of some of them are given in Appendix 1.

5.5. Simple media with unspecified additives There are a number of media available which are not based on a detailed investigation of growth requirements, but rather include crude mixtures of nutrients added to promote cell growth. These include lactalbumin hydrolysate (Appendix 1: Table 9) or yeast extract (Appendix 4) to provide an inexpensive source of amino acids or vitamins. Thus Melnick’s monkey kidney media A and B (Melnick, 1955) contain lactalbumin hydrolysate and calf serum in Hanks’ and Earle’s BSS, respectively. Chick embryo extract and tryptose phosphate broth (Appendix 1, Tables 11 and 12) are also used occasionally and their use is referred to where appropriate throughout the book. Mitsuhashi and Maramorosch mosquito cell medium contains lactalbumin hydrolysate, yeast extract and foetal calf serum in a specially developed saline (Mitsuhashi and Maramorosch, 1964; Singh, 1967).

5.6. Antibiotics The method of preparation of antibiotic stock stolutions for use in cell culture media is given in Appendix 1, Table 6. It is more usual,

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however, to buy sterile solutions at X 50 or X 100 from commercial suppliers. It should be stressed that the use of antibiotics in cell cultures should be restricted to short-term cultures, and routine subculturing of cell lines should be done in the absence of antibiotics so that the selection for antibiotic-resistant strains of bacteria is discouraged. Apart from encouraging sloppy technique, antibiotics can be used to ‘cure’ a special culture should it become infected (see for example 0 9.9). This, of course, if only possible if the culture is initially growing in antibiotic-free medium and it is not a recommended procedure if it can be avoided. Most antibiotics have a limited stability and only remain active for 3-5 days at 37°C. Many are toxic to cells and so should only be used at the recommended concentration. Table 5.1 lists the antibiotics which have been used in cell culture media. The first six are most commonly used. These are frequently available from supplies as mixtures, active against a wide range of contaminants.

5.7. Serum Serum supplies many essential factors which can be classified as follows (see 0 2.4 and 2.5): a) adhesion promoting components necessary for adhesion and spreading of cells (Yamada and Olden, 1978), b) nutrients and trace minerals (Shipley and Ham, 1981), c) transport proteins, e.g. transferrin and albumin. d) growth factors and hormones, and e) protein e.g. albumin or fetuin which may act non-specifically to bind toxic substances and stabilise labile components. Plasma cannot usually replace serum as it lacks PDGF released from platelets on clotting. Despite attempts to grow cells in fully synthetic media, the vast majority of cells are still grown in media containing some natural additive, usually serum. Although a variety of sera are commercially available, most cells grow best in the presence of bovine serum. This

TABLE 5.1 Antibiotics in use in cell culture media

Antibiotic

Recommended concentration

1) Penicillin-G 2) Streptomycin sulphate 3) Amphotericin-B (Fungizone) 4) Kanarnycin 5 ) Gentamycin 6 ) Nystatin (Mycostatin) 7) Neomycin 8) Chlortetracycline 9) Tylosin tartrate 10) Chloramphenicol 11) Erythromycin 12) Polymixin B ? Indicates confusion in the literature.

Gram ve bact

+

Gram - ve bact

Yeast

Fungi

Moulds

+

+

+

+

+

Mycoplasma

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is obtained either from the foetus or the newborn calf (foetal bovine or calf serum). Obtaining serum (especially foetal bovine serum), processing and sterilising it is a difficult procedure requiring special apparatus and is best left to the commercial companies, e.g. Flow Laboratories, Gibco etc. (see Appendix 3). It is important that haemolysis is kept to a minimum and only suitable batches of serum are processed. Processing involves passage through a series of filters down to O.lprn, after which the product undergoes a series of tests to detect possible bacterial, mycoplasmal or viral contamination. In addition, each batch of serum is tested to ensure that it will support the growth of primary, diploid and established cell lines, using both serial passage and plating efficiency tests. Before buying serum (which is an expensive item) the customer is recommended to obtain samples of various batches and to repeat a plating efficiency test (0 7.1.1) or to measure the growth rates and yields of at least two cell types. It is particularly important that the batch of serum supports the growth of the cells in the customer’s laboratory. Most commerical companies are in favour of this trial and moreover will retain supplies of a particular batch of serum in their own -20°C store, delivering a fraction of the order each month. Companies are also prepared to provide serum from a particular herd of cows (donor serum) which ensures a greater degree of batch to batch consistency. Once prepared, serum should be stored at -20°C at which temperature it remains stable for 6 months or more. It should be thawed slowly (by standing at 4°C) and not be subjected to repeated cycles of freezing and thawing. Serum contains complement which may interfere in virus or cytotoxicity assays. This can be inactivated by heating the serum to 56°C for 30 min but this may also destroy some growth factors. 5.7.1, Removal of small molecules from serum

This is essential for nutritional studies and is often desirable when labelling cultures with certan radioisotopes, so that (a) the specific activity of the precursor may be accurately known, and (b) the specific activity may be the maximum possible. Thus, it is common

83

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CH. 5.

to label cells with a tritiated amino acid to follow the rate of protein synthesis, and so as to maximise incorporation, special medium is made lacking that amino acid. Serum may contain, however, a significant amount of that amino acid and the best results will only be obtained if this is first removed. Two methods are available. 1) Sephadex treatment. A sterile column (2.5 X 100 cm) containing about 50 g dry weight Sephadex G-50 (coarse) is well washed with 5 1 sterile 0.85% saline. The Sephadex slurry may be sterilised by autoclaving for 15 min at 121°C before packing. 200 ml of whole serum may be applied to such a column and eluted with saline at 4-5 ml/min. The excluded fraction elutes between 200 and 400 ml. TABLE 5.2 Material removed from serum by gel filtration or dialysis Constituent

Whole serum

Sephadexed serum

Alanine (mM) Arginine (mM) Aspartic acid (mM) Glycine (mM) Glutamic acid (mM) Histidine (mM) Isoleucine (mM) Leucine (mM) Lysine (mM) Methionine (mM) Phenylalanine (mM) Proline (mM) Serine (mM) Threonine (mM) Tyrosine (mM) Valine (mM) Total protein (gW) Cholesterol (mgW) Uric acid (mgW) Glucose (mgW) Potassium (mEq/l) Calcium (mgW) Alkaline phosphatase (units)

0.672 0.246 0.146 0.408 0.309 0.216 0.121 0.347 0.129 0.015 0.126 0.387 0.295 0.261 0.498 0.336 7.3 135 1.4 132 5.8 7.2

0 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0 0 0 0 < 0.001 < 0.001 < 0.001 0 0 6.2 113 0.8 6 0 2.8

< 0.001 < 0.001 < 0.001 < 0.001 0 0 < 0.001 0.005 0.001 0 < 0.001 < 0.001 0 0 6.5 122 0.7 7 0 0.2

15.9

13.2

14.0

0

Dialysed serum

< 0.001


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CELL CULTURE FOR BIOCHEMISTS

2) Dialysis. Dialysis tubing, sterilised by boiling in distilled water, may be filled with serum, using aseptic conditions, and dialysed against 2 changes of 100 volumes of sterile saline or BSS at 4°C over 48 h. At the end of this time the serum may be removed from the sac again using aseptic conditions. Table 5.1 is taken from Patterson and Maxwell (1973) and gives the composition of serum before and after treatment. Only limited proteolysis occurs over a 3-week period even at 37”C, but it is not recommended that such serum be stored for long times, otherwise small molecules may be released. Serum also contains steroid hormones and, in experiments where these are the object of study, they can be removed by treatment of the serum with dextran-coated charcoal, as follows:Prepare dextran-coated charcoal by stirring 2.5% (w/v) Norit-A charcoal and dextran T-70 (0.0258 w/v) in PBS-A for 18 h at 4°C. Centrifuge 1000 g for 5 min at 4°C and resuspend the pellet in heat inactivated serum. Stir the suspension for 18 h at 4°C and then resediment the charcoal. Pass the stripped serum through a prefilter and a 0.45pm filter before sterilising by filtration through an 0.2pm filter (see 0 8.3). Store at - 20°C (Table 5.2).

5.8. Serum-free media Certain advantages are perceived in being able to grow cells in a completely defined medium. Thus it becomes possible to identify material appearing during cultivation and to study the effects of trace materials (e.g. hormones and growth factors) on cells in the absence of interference from substances present in serum. This situation may be far from normal, however, and no defined medium remains so for long as it quickly becomes ‘conditioned’ by the growth of cells. Two major disadvantages in the use of serum (lack of reproducibility in quality and possible risk of contamination) are avoided by using serum-free medium. In addition, it is possible to culture a wider variety of differentiated cells using defined media.

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CH. 5.

TABLE 5.3 Growth with various commercial serum-free media and media containing serum extenders Basal medium RPMI1640 RPMI 1640 RPMI1640 RPMI 1640 RPMI 1640 RPMI 1640 RPMI 1640 Hybri-clone SF Gibco-high protein Gibco-low protein SF-1 SF-1 SF-1

Factor added

% Growth

0.1% ESG 1% ESG 1%SF-M 1%Nutridoma 0.1% Mito+ 10%CPSR-3 -

(0% FBS) 5 4 3 0 9 9 43 0

* (2% FBS) 32 23 37 62 85

100 73

cost (E/I) (0%FBS) (2% FBS) 4.50 9.70 9.30 14.50 52.50 57.70 40.50 45.70 24.50 29.70 15.10 20.30 25.00 110.00 115.20

-

11

ND

35.00

-

2 141 157 143

ND 160 145 161

35.00 25.00 29.80 73.00

-

0.1% ESG 1%ESG

30.20 35.00 78.20

* Growth expressed as a % of that obtained with RPMI 1640 plus 10%FBS. Nutridoma SP is supplied by BCL as a X 100 concentrate. Mito + is supplied by Collaborative Research Products through Flow Laboratories Limid., as a X lo00 concentrate. Hybri-Clone SF is supplied by ICN as a X l medium or as a XlOO concentrate (SF-M) to be added to normal medium. Gibco BRL sell high and low serum-free hybridoma media ( X 1). SF-1 is a serum-free medium ( x 1) supplied by Costar through Northumbria Biologicals Ltd. and which can be supplemented with Ewing’s Sarcoma Growth Factor (ESG). CPSR-3 (Controlled process serum replacement) is an adult bovine serum modified for growth of hybridoma cells. The costs of RPMI and FBS reflect our bulk purchase agreements and are somewhat below catalogue prices.

It is also claimed that serum-free media are cheaper, but this is not generally my experience as seen in Table 5.3. One of the major problems in establishing cultures of epithelial cells is the overgrowth of these cells by the faster growing fibroblasts and serum-free media have been devised which do not permit fibroblast growth, thus allowing cultures of mouse kidney epithelial cells to be obtained (Taub et al., 1979; Taub and Sato, 1980) (Fig.

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87

5.2). Different selective media may enhance growth of different sorts of epithelial cells from such cultures (Chung et al., 1982) but the differentiated functions are rapidly lost if serum is added to the medium (Lieberman and Ove, 1958; Becker and Willis, 1979). The selectivity of particular defined media can be a disadvantage in that each individual cell type requires a different defined media. N.B. Fibroblasts tend to lack D-amino acid oxidase and replacement of L-valine with D-valine in media encourages the growth of epithelial cells. Thus the aims of those using serum-free media are 3-fold: 1) To reduce the cost of using FBS. 2) To have a culture supernatant free (or partly free) of protein. 3) To have a defined medium for studies of growth factors or hormones.

5.8.1. Low serum media

In order to reduce costs the concentration of foetal bovine serum (FBS) can be reduced in growth media. Figure 5.3 shows the effect of lowering the serum concentration on the growth of a rat hybridoma cell line. Initially cells can be plated in 5 % FBS and later this concentration can be reduced by using a combination of FBS (1.5%) and new born calf serum. This leads to yields of diploid fibroblasts on Cytodex-1 microcarriers of 80% those obtained with 10% FBS (Clarke, 1983) (0 3.6). Further economies can be obtained by using 0.5% FBS together with BSA (2 mg/ml) and EGF (10 ng/ml) which gives a yield of 85% that obtained with 10% FBS. However, attachment requires a period of growth in 5% FBS indicating this as a major function of Fig. 5.2. Primary baby mouse kidney cultures were established at about lo3cells/cm2 in medium based on a 50: 50 mixture of DMEM : F12 supplemented with 10% FBS (a) or a mixture of 5 hormones (PGE1, hydrocortisone, triodothyronine, insulin and transferrin (b). Although over 99% of the attached cells were epithelial at day 1, by the time the photograph was taken (day l l ) , fibroblasts had completely overgrown the epithelial cells in the serum-supplemented medium. Only epithelial cells are present in the hormone-supplemented culture. (Reproducedfrom Taub et al., 1979, with thanks.)

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serum. This requirement can be largely circumvented by the use of fibronectin (2pg/ml) but insulin (lpg/ml) and transferrin (25pg/ml) are then necessary for continued growth and fetuin (1 mg/ml), dexamethasone (50 ng/ml) and putrescine (1OOpM) increase the yield, as do the trace metals molybdenum (0.5 nM ammonium molybdate), cadmium (50 nM CdSO,) and selenium (15 nM H,SeO,). As the medium becomes more defined the costs do rise, however. 5.8.2. Defined media

Which defined medium is the best depends on the cell type being studied. R.G. Ham set out to obtain defined media for different cell types. Initially designed for diploid human fibroblasts his group devised a series of media at the Department of Molecular, Cellular and Developmental Biology of the University of Colorado (MCDB media - see Appendix 1) which support optimal growth when supplemented with 2% dialysed calf serum (Ham et al., 1977; Ham, 1982). The serum can be replaced with insulin (0.95pg/ml) dexamethasone (1.4pM), EGF (30 ng/ml), a lipid mixture (lOpg/ml) and prostaglandins and reducing agents but this is accompanied by a 40% drop in clonal growth (Ham, 1983). Most of the supplements of serum-free medium can be isolated from serum, but more recently other additions have been made which have allowed culture of some differentiated cells which could not previously be cultured in vitro. In these experiments Iscove’s medium or a 1/1 mixture of DMEM and Hams F12 or RPMI1640 is used as the basal medium, and this is supplemented with different substances for different cells. Thus for RF1 rat ovarian cells insulin (2pg/ml), transferrin (25pg/ml), hydrocoritsone (10 nM) and fibronectin (8pg/ml) are added; while for MCF-7 cells insulin (O.lpg/ml), transfemn (25pg/ml), prostaglandin F2 (100 ng/ml), fibronectin (8pg/ml), EGF (100ng/ml) and serum spreading factor are required for cells to show oestrogen-dependent growth (Barnes, 1985). Hela cells can be grown with added transfemn (5pg/ml), insulin (Spg/ml), EGF (10 ng/ml) and hydrocortisone (50 nM).

CH. S.

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89

These examples illustrate the work that is required to obtain a defined medium for a particular cell type. Many other examples are given in Taub (1985), and it is clear that to obtain optimal growth a systematic approach is required for each individual cell line. This is obviously not possible to achieve in most cases but commercial suppliers now provide a series of serum-free media which support the growth of many cell types. Some of these are listed in Table 5.3. Although individual cell types may grow in a series of defined media they may still require the presence of particular growth factors or hormones to express a differentiated phenotype. In the presence of serum some cells take on new properties. Thus certain adrenergic neurons begin to respond also to acetyl choline i.e. they take on a dual function. This change also occurs if serum is replaced by conditioned medium from heart cells (Patterson, 1978), but growth in serum-free media precludes this change (Iacovitti et al., 1982). The opposite effect is, however, sometimes observed. Thus the MCF-7 human breast cancer cell line only exhibits oestrogen dependent stimulation of growth in the presence of high levels (15%) of FBS which is presumed to provide a factor which is otherwise rate limiting for oestrogen-dependent growth (Dembinski and Green, 1983, and see below).

5.8.3.Media for isolation of secreted products A major stimulus to the development of serum-free media has been the desire to be able to obtain immunoglobulins secreted by hybridoma cells, simply by sedimenting the cells. However, low-protein media are not as effective as those containing higher levels of protein unless a significant period of adaptation is allowed. Because of the low protein content of some serum-free media the cells may be more fragile and fibroblasts and epithelial cells may attach only poorly to the substratum. In such cases it is important to adapt cells to growth in serum-free medium by growing cells in steadily decreasing concentrations of serum. The following protocol is adapted from information supplied by Boehringer Company Limited for adaption of cells for growth on 1%Nutridoma:

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CELL CULTURE FOR BIOCHEMISTS

1) Plate cells out in triplicate at lo5 per ml basal medium (usually a 1 : 1 mixture of DMEM and RPMI 1640) plus 1% Nutridoma containing 10, 7.5, 5. 2.5 and 0%serum. 2) After 3-5 days count the cells and select those in the lowest serum concentration that have multiplied 10 fold. This is unlikely to be 0% serum, but in my experience it is likely to be those in 2.5% serum. 3) Replate these, as above, in medium containing 1% Nutridoma plus 2, 1, 0.5, 0.2 and 0%serum. 4) Repeat this process until the cells are adapted to growth in 0% serum and maintain them without serum for 4-5 passages to ensure stability. N.B. Cells cannot be frozen in protein free medium and will require adaptation each time a batch is thawed (see 0 7.3). It is also not recommended to use protein free medium for cloning unless it is supplemented with factors such as ESG and/or HECS. A serum-free medium supplemented with insulin, transferrin, ethanolamine and selenium (ITES) allows growth of certain hybridomas at 17-74% the rate found with 15% FBS (Wolpe, 1984); and Cleveland et al. (1983) devised a protein-free medium for growth of myeloma cells which, with addition of BSA at 2.5 mg/ml, forms the basis of Costar’s SF-1 and SF-X supplemented media. Cloning is still very difficult in serum-free media, but feeder layers can be replaced by culture supernatants from human endothelial cells (HECS; Astaldi, 1983) or Ewing’s sarcoma cells (ESG; Ley et al., 1980) - see 6 5.8.5. An easier (and more economical) way of isolating immunoglobulins or other secreted materials is to grow the cells to high density in medium containing serum and then transfer them to serum-free medium for 2 or 3 days. Production and secretion continues for this time and the antibodies are more easily purified from the cell supernatant (see also 8 5.8.5). The cells can then be diluted into serum-containing medium as a seed for a new production run, though this should not be repeated too often as mutant clones may develop.

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91

5-

g

4-

c

aJ

n

s

3-

-0

u

2-

1-

'I0

FBS

Fig. 5.3. Rat hybridoma cells growing in RPMI containing different concentrations of FBS. 180,ooO cells were inoculated into 25 cm2 flasks in 5.5 ml RPMI containing the indicated concentration of FBS. The cells had previously been maintained in 10% FBS. Cells were counted after 3 days. The doubling time for the cells growing in 10% FBS was 14.7 h.

5.8.4. Commercial media

I have tested a limited number of commercially available serum-free media and serum extenders for growth of a rat hybridoma cell line. The cells are normally grown in RPMl 1640 with 10% FBS and 180,000 cells were transferred directly, without adaptation, into 5.5 ml test medium in a 25 cm2 flask. The cells were counted on day 3 when the controls had grown to 5.2 million (Fig. 5.3). It is clear that an immediate saving could be made by growing the cells in 5% FBS which gave over 90% of the growth obtained in medium containing 10%FBS. Table 5.3 shows growth in a series of different media with or without 2% FBS. None of the supplements tested gave maximum growth in the absence of serum but Mito+ and Nutridoma gave growth similar to control values in the presence of 2% FBS, i.e. true

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serum extenders; and Mito + is also a money saver as the cost per litre is only €20.30 compared with €30.50 for RMPI 1640 10% FBS. Costar’s SF-1 (Appendix 1) gave excellent growth even in the absence of serum or ESG and was also an economic proposition. There was a change in cellular morphology, however, with the cells growing in quite large floating clumps. Some of the serum-free media were ineffective and some were very expensive. Better results may be obtained after a period of adaptation: after 3 days adaptation in 2% serum, cells were transferred to serum-free medium. Those in Mito + or in Costar’s SF-1 continued to grow, albeit at a reduced rate, for several weeks providing a culture supernatant from which monoclonal IgG could be harvested. Although serum-free many of the media are not protein-free. For example Costar’s SF-1 contains 2.5 mg BSA per ml compared with the value of 3.5 mg protein/ml of RPMl 1640 + 10% FBS. It is free of antibodies, however. Medium containing 0.1% Mito gave the best growth at the lowest protein concentration. Sigma Chemical Company’s CPSRs are adult bovine serum modified to give a consistent serum, low in endotoxin and y-globulin and supplemented with factors so as to resemble foetal serum. It is clear that some of the media are very expensive, and care must be taken to note the expiry date, which is never far in the future (and was over a year in the past when delivered, in one instance). These results cannot necessarily be extrapolated to other cells as Wolfe (1984) has shown that different cells grow at vastly different rates. However, faster growing cells seem to be faster growing in several different media he tested. New serum-free or low serum media are appearing regularly and it is advisable to write to the various companies to obtain the most up-to-date information.

+

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5.8.5. Isolation of factors from culture supernatants

Factors such as multiplication stimulatory activity (MSA) are produced by foetal rat liver explants and can be obtained from conditioned medium as follows (Nissley et al., 1979):

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1) Grow Buffalo rat liver (BRL) cells to confluence in Ham's F12 or DMEM containing 58 FBS. 2) Rinse in medium lacking serum and continue to incubate the confluent monolayer of cells in serum-free medium. 3) MSA accumulates in the medium reaching a plateau after 3 days. In a similar way, sarcoma growth factors (SGFs e.g. ESG) can be obtained by the serum-free cultivation of murine sarcoma virus transformed 3T3 cells or transformed rat kidney cells. Nerve growth factor (NGF) is produced in excess by the human melanoma cell line A375 and fibroblast growth factor (FGF) is secreted by fibrosarcoma cells (Todaro et al., 1979). A 50 : 50 mixture of fresh growth medium with one of the above conditioned media is often recommended for growth of embryonic stem cell cultures. Otherwise the protein fraction can be concentrated for addition to serum-free medium. In contrast small amounts of HECS (human endothelial cell supernatant) will supplement serum-free media for the growth of hybridomas. HECS is the culture supernatant from cells grown in the presence of 308 pooled human serum. The important factors are only released from the endothelial cells in the presence of the human serum, but continue to be secreted for 3 days (Astaldi, 1983).

5.9. Media for culture of insect cells The aim in designing a culture medium is to mimic to some degree the composition of the fluid surrounding the cells in vivo. This proved difficult for insect cells in the 1930s as the composition of haemolymph was unknown and has been shown more recently to be less important than originally considered (Yunker et al., 1967; Vaughan, 1971). Thus, although the inorganic salt concentration of insect culture medium may reflect that of haemolymph the amino acid and vitamin component may be supplied by lactalbumin hydrolysate, whole egg ultrafiltrate and bovine serum when haemolymph is not available. Foetal bovine serum proved to be the most effective of several sera tested (Mitsuhashi and Maramorosch, 1964; Sing,

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1967). The high levels of amino acids found in haemolymph may be supplied from a 10% lactalbumin hydrolysate supplemented with non-essential amino acids. Few studies have been done on the amino acid requirements of cultured insect cells and, although some media (Grace, 1962) do specify an amino acid mixture, the presence of additives, such as whole egg ultrafiltrate, makes the significance of specific amino acid concentrations doubtful. Glucose is the usual carbohydrate for energy source in culture even though in vivo a variety of carbohydrates are found in haemolymph (Wyatt and Kalf, 1957). The B vitamins and ascorbic acid are considered essential (Samborn and Haskell, 1961). The inorganic constituents vary widely between one insect culture medium and another. The pH is generally just below 7, but even this is not always optimal as Aedes albopictus cells are cultured at pH 7.2.

5.10. Media for culture of plant cells Plant cells do not have the demanding requirements of animal cells and do not require the complex basal media described earlier in this chapter. Rather they manage with a mixture of inorganic salts, trace elements and sucrose, presented either as a liquid medium or as a solid, agar-based substratum. Seldom are amino acids other than glycine required, though sometimes a casein hydrolysate (casamino acids) or yeast extract is provided and this may act as a supply of organic nitrogen. Some vitamins are found to improve growth (see Appendix 1). Often coconut milk or coconut water is supplied. The latter is obtained as the ‘whey’ when coconut milk is autoclaved and the precipitated protein removed by filtration. Apart from poorly defined nutrients, coconut milk provides the growth factor, kinetin, but it is preferable to supply this factor separately and avoid the use of the coconut milk, which varies greatly from batch to batch. The most common media are those of Murashige and Skoog (1962), and Gamborg et al. (1968), which are available in powder form from suppliers such as Flow Laboratories or Gibco. The

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composition of the latter is given in Appendix 1: Table 22. They are reconstituted as follows: 1) Add the powder slowly, with stirring to about 80% of the final volume of water. 2) Adjust the pH with 1N NaOH or HC1 to pH 5.0 for liquid culture or pH 5.7 for an agar-based medium. 3) Add agar, if required, and dissolve by heating gently to boiling with continuous mixing. 4) Dispense into appropriate containers (e.g. cotton wool plugged conical flasks) and autoclave at 15 p.s.i. for 15 min. 5) Cool and store at room temperature or 4°C.

Callus cultures, initiated from leaves or stems of growing plants are grown on an agar based medium in the presence of growth factors (cytokinins and auxins, e.g. 2,4D or IAA). Undifferentiated cells can also be grown in suspension in similar media (without agar), but to initiate differentiation the concentration of auxin must be lowered. The optimum concentration of hormones must be determined by experimentation for each cell type. More details are to be found in Dixon (1985).

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CHAPTER 6

Primary cells 6.1. Introduction Cells taken from an animal and placed in culture are termed primary cells until they are subcultured. This chapter is concerned with the methods used to obtain cells from animals. Only a limited number of examples are given and there are numerous specialised texts concerned with individual tissues. As far as humans are concerned it is important to obtain cells while bringing about minimum discomfort to the donor. For this purpose two cell types are commonly used: they are the lymphocyte and the skin fibroblasts but cells of the luminal epithelium can be obtained from milk. The lymphocyte is in general a non-dividing cell and requires mitogenic stimulation, usually by phytohaemagglutinin (PHA) or other plant lectin, when it undergoes at most a few cell divisions before dying. The skin fibroblast also has a limited, but far longer, lifespan (Hayflick, 1965a) but is seldom available in the same quantity as the lymphocyte. Only a small fraction of the cells in a preparation obtained from a tissue will go on to divide. For this reason, seeding levels are often high and microcarriers are not very suitable initially as many may remain without a dividing cell attached.

6.2. Lymphocytes Lymphocytes have been isolated from a variety of mammalian sources. The use of pig lymphocytes (Forsdyke, 1968) usually involves slaughter of the animal, but the horse is an ideal source if a large (200 ml) regular supply of lymphocytes from one donor is 91

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required (Zain et al., 1973). Human lymphocytes are an ideal source of human tissue for the study of genetic diseases (Abo-Darub et al., 1978). It should be noted that human blood is a potential source of human immunodeficiency virus (HIV) and hepatitis B virus, and it and all waste material should be disposed of accordingly. Human blood samples should only be taken by qualified staff under conditions guaranteed to protect patient and scientist from risk. 6.2.1. Isolation of leukocytes and autologow plasma

Blood is taken aseptically into a flask containing heparin (2500 I.U./lOO ml blood). ‘Pularin’ (Evans Medical Co.; Appendix 3) is a suitable source of heparin without preservative. The blood is allowed to stand at room temperature for 40 min and the leukocyte-rich plasma is then withdrawn aseptically and centrifuged at 400 g for 15 min at room temperature. The supernatant is withdrawn and recentrifuged at lo00 g for 10 min at room temperature to give a cell free autologous plasma preparation which can be stored at - 20°C. The cell sediment is resuspended in l/lOth the initial volume of autologous plasma. 6.2.2. Purification of lymphocytes

Two different methods are in general use: the glass bead column method of Rabinowitz (1964, 1973) and the density gradient centrifugation method of Boyum (1968). 6.2.3. Glass-bead column method

A sterile glass condenser or water jacketed column (40 X 2 cm) is clamped in a vertical position and water at 37OC is circulated around it. This size of column is large enough to fractionate leukocytes from 200 ml of blood. Glass beads (Type 070-5005 superbrite brand from 3 M Company, St. Paul, Minnesota) are soaked overnight in concentrated HNO, and then rinsed exhaustively in tap and distilled water before

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drying and sterilising at 160°C for 3 h. The sterile beads are poured into the column to fill it 3/4 full and then washed with Eagle’s MEM (pregassed with 5% CO,). The leukocyte suspension is applied to the top of the column and allowed to percolate in. Considerable losses may occur at this stage as the cells stick to the tubes and pipettes, but this can be countered by using siliconised glassware (e.g. treated with Repelcote; Appendix 3). The cells are left on the column at 37°C for 30 min and then eluted with Eagle’s MEM supplemented with 50% autologous plasma. Many of the lymphocytes and any erythrocytes are eluted in the first column volume, closely followed by the platelets and usually the first 40 ml is collected from a 40 X 2 cm column. Granulocytes and monocytes are only eluted with 0.02% EDTA in PBS-A. Failure of this method may be associated with the use of aged serum or plasma and hence the fresh autologous plasma is recommended. If only limited amounts of this are available its concentration may be reduced. The contaminating red blood cells may be removed by hypotonic lysis but this sometimes leads to destruction of some lymphocytes (Dain and Hall, 1967;Roos and Loos, 1970). 6.2.4. Gradient centrifugation method

Triosil (Na-metrizoate or Na-N-methyl-3,5-diacetamido-2,4,4-triodobenzoate) is provided as a radiographic contrast medium (60% (w/v) solution containing 55.2% (w/v) Na-metrizoate, 2.8% (w/v) Ca-metrizoate and 2% (w/v) Mg-metrizoate) by Glaxo Laboratories Ltd. (Appendix 3). The density of this solution is 1.39 g/ml and it is diluted with water to 1.2 g/ml. Just before use 10 parts are further diluted with 24 part 9% (w/v) Ficoll (Pharmacia Chemicals, Uppsala; aqueous solution sterilised by autoclaving) to give ‘separation fluid’; ready prepared mixtures; ‘Ficoll-Paque’ and Histopaque are available from Pharmacia and Sigma (Appendix 3). 10 ml of leukocyte suspension are diluted with 20 ml sterile 0.9% NaCl and 28 ml layered over 10.5 ml of separation fluid in a sterile 14 x 3 cm tube. Smaller quantitites may be used but the height of the layers should be approximately the same. After centrifugation at 400 g for 25 min at 20°C the red cells are clumped by Ficoll and sediment to the bottom of the tube while the lymphocytes form a

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white band at the junction of the separation fluid and saline layers. This is removed with a syringe and the cells are washed twice by resuspension in Eagle’s MEM (Appendix 1) supplemented with serine (10 pg/ml) and glycine (7.5 pg/ml), and centrifugation at 800 g for 10 min. The cells are finally resuspended in this modified Eagle’s MEM containing 10% autologous plasma. A differential white blood cell count (Hunter and Bomford, 1968) shows that 80-90% of the cells are small lymphocytes. A more direct method recommended by Pharmacia (Appendix 3) uses 2 ml whole anticoagulant-treated blood diluted with an equal volume of BSS. This is layered over 3 ml ‘Ficoll-Paque’ and sedimented at 400 g for 30 min. The lymphocytes may be removed from the interphase and the upper layer may be used as a source of autologous plasma. An alternative dense medium for fractionating blood cells is ‘Percoll’ also provided by Pharmacia (Appendix 3). Percoll consists of particles of colloidal silica coated with polyvinylpyrrolidone (Pertoft et al., 1978). It is made up by dilution into isotonic saline and forms a self-generating gradient on centrifugation at 20,000 g for 15-20 min in an angle rotor. Onto this gradient may be layered a sample of anticoagulant-treated blood which can be fractionated in two stages. The first stage involves centrifuging for 5 min at 400 g and removing the plasma layer containing the platelets from the top of the gradient. The second stage involves centrifuging for 15 min at 800 g when lvmphocytes, granulocytes and erythrocytes band at their isopycnic densities. They can be harvested by upward displacement using 60% sucrose and diluted and washed with saline solutions by sedimentation at 400g. Various methods are available for fractionating lymphocytes to obtain pure cultures of the different sorts of white blood cells (Johnstone and Thorpe, 1987). A particularly simple method involves the use of magnetisable polymer particles precoated with monoclonal antibodies (Dynal Ltd.; Appendix 3). 6.2.5. Cultured lymphocytes

The cells, suspended at lo6 per ml of Eagle’s MEM supplemented with serine and glycine and 10%autologous serum and containing 50

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I.U. of penicillin and streptomycin per ml, are added to 12 X 1.3 cm capped tubes (1 ml), 6 cm Petri dishes (2 ml), 14 x 3 cm tubes ( 5 ml) or roller bottles (50 ml upwards). The vessels are either loosely capped and incubated in a CO, incubator or are gassed with 5 % CO, in air and sealed prior to incubation. To induce blastogenesis an equal volume of medium containing mitogen is added. The most commonly used mitogen is PHA. 100 mg PHA-M (Difco Laboratories; Appendix 3) is rehydrated with 5 ml sterile distilled water and further diluted 1 in 200 with growth medium. Pokeweed mitogen (Gibco Biocult, Ltd.) is reconstituted in a similar manner but only diluted 1 in 40 with growth medium to give an active solution.

h a f t e r PHA addition

Fig. 6.1. DNA synthesis in PHA-stimulated horse lymphocytes. Aliquots of 3X106 horse lymphocytes purified by the glass bead column method were incubated for various lengths of time with PHA-M in tubes in 3 ml Eagle's MEM (supplemented with 10 pg serine/ml and 7.5 pg glycine/ml and 10% autologous horse plasma). Before harvesting the cells were labelled for 6 h with ['4C]thymidine (3.66 Ci/mol; 1 pCi/ml). 0 , Amethopterin (10-6M) was added 16 h before harvesting. 0, no amethopterin added. (Courtesy of Dr. B.S. Zain, 1971.)

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Immediately on adding mitogen the cells begin to clump and it is for this reason that is preferable first to dispense the cells into their individual vessels as reproducible aliquots are not easily obtained once the cells have clumped. Under these conditions DNA synthesis - as measured by incorporation of radioactive thymidine into acidand ethanol-insoluble material (see § 12.1) - starts around 24 h after mitogen addition and increases in rate over the next 2 or 3 days. This labelling of DNA may be for 6 h periods with [14C]thymidine(2 pCi/ml [2-14C]thymidine,3.7 Ci/mol) or for 2 h periods with [3H]thymidine ([6-3H]thymidine at 4 pM) after which the cells are sedimented, washed three times with 5% trichloracetic acid, twice with absolute ethanol, dried with ether and solubilised in sodium hydroxide for counting in a scintillator such as Ecoscint (National Diagnostics; Appendix 3). The sensitivity of the assay may be increased by causing partial synchrony of DNA synthesis. This may be achieved by pretreatment of the cells with amethopterin (5 X lO-’M) for several hours before and during the exposure to radioactive thymidine (Tormey and Mueller, 1965; Pegoraro and Benzio, 1971) (see Fig. 6.1).

6.3. Human skin biopsies -

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Select an area of skin - usually on the inner side of the upper arm - which is free of hair and minimally keratinised. Sterilise by swabbing with 70% ethanol and pull a piece of skin up with forceps. With sharp scissors excise 1 mm3 of the skin and place in a 6 cm Petri dish. Add a drop of complete growth medium (e.g. Dulbecco’s MEM containing 10% foetal calf serum) and cut the skin fragment into 5-10 pieces. Dispose the several pieces over the surface of the dish and cover each with a drop of complete medium. Incubate overnight at 37°C. Sometimes this incubation if done with the dish tilted so that the medium rests at the edge of the

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Fig. 6.2. Primary cells growing out of human skin explant. Seven days after fragments of human skin are placed in culture fibroblasts and epithelial cells can be seen growing out of the explant. (Courtesy of Dr. M.B. Hodgins.)

dish thereby maintaining high humidity yet encouraging attachment of the tissue fragments to the surface of the dish. - Very carefully, so as not to dislodge the fragments, add 3 ml complete medium to the dish and continue the incubation. Alternatively, rather than using dishes, the tissue fragments may be transferred to a small bottle and 5 ml complete medium may be added in such a way that the bottle can be incubated with the fragments adhering to the underside of the upper surface. When the fragments have attached after 16 h the bottle is turned carefully so that the medium now bathes the fragments. Fibroblasts grow out from the tissue fragments within a week (Fig. 6.2).

6.4. Mouse or rat embryo cultures -

Kill pregnant (at least half-term) mice by breaking the neck.

- Immerse the mice in methylated spirits and place under a UV

light for 5 min to sterilise them exteriorly.

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Aseptically, using forceps and scissors, cut through the skin under the forelegs and strip it off down to the rear legs. Using fresh forceps and scissors, open the abdomen and remove the uterus into a large Petri dish. Rinse each uterus with 25 ml of PBS-A containing 1% penicillin/s treptomycin. Remove the embryos with fresh forceps and scissors into warm PBS-A. Agitate and discard the PBS-A. Wash until clear. Check after each wash (using brain heart and Saboraud medium) that the embryos are contamination-free. Remove the viscera if required and place the embryos into sterile universals (about six mouse embryos per bottle). Mince the embryos with scissors and wash with PBS-A until clear. Check for contamination as before after each wash. Add warm 0.25% trypsin in PBS-A (about 5 ml per embryo) to the mince and transfer to a 500 ml dimpled flask. Add a sterile teflon or silicone rubber covered magnetic stirrer. Stir gently at 37°C for 15 min. Remove from the magnetic stirrer and allow the tissue to settle. Remove trypsinised cell suspension, i.e. non-settled material, to a 250 ml centrifuge bottle containing 20 ml calf serum. Add fresh trypsin to the settled material. Repeat the trypsinisation once or twice, checking each suspension for contamination. Centrifuge the cell suspensions at 150 g for 5 min and discard the supernatant fluids. Resuspend the cells in a small volume of warm PBS and then fill the bottles with PBS. Allow large particles to settle to the bottom of the bottle. Pipette off any froth and floating particles. Filter the cell suspension through a sterile gauze filter to remove any clumps. Centrifuge at 150 g for 5 min. Seed 75 cm2 flasks with 40-50 X lo6 cells in 50 ml ETC (Appendix 6). Seed 8002 roller bottles with 2 X lo8 cells in 200 ml ETC (Appendix 6).

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Gas with 5 % CO, (see 0 3.1.2) and incubate at 37°C for 2-5 days. Then check for sterility. - Subculture by splitting 1 to 2 or 1 to 3. -

Alternatively, the subconfluent cultures can be maintained at 3OoC for several weeks if the medium is replaced twice a week. The day before the cells are required, they should be returned to 37°C.

6.5. Chick embryo cells After a 10-day incubation period remove the eggs from the incubator and candle to determine viable embryos. Only well-developed active embryos with good blood supply are used. - Leave under UV light for 10 min with the air sac end uppermost. Swab the entire egg with 70% alcohol. - Crack the shell over the air sac and remove pieces of shell with sterile forceps. - Cut away the shell membrane and chorioallantoic membrane using sterile forceps. - With a pair of curved forceps, remove the embryo by the neck into a 90 mm Petri dish. - 'Remove head (internal organs, e.g. liver may also be removed and processed separately; see below) and transfer the carcass to a conical flask (100 ml) of warm PBS-A (Appendix 1). - Use fresh forceps and scissors for each egg or carefully sterilise the instruments by flaming. - Swirl the embryos in PBS-A to wash off adherent blood, and discard PBS-A. - Mince the embryos as finely as possible by chopping with scissors either in the conical flask or in a universal container (mince may be transferred in PBS-A if necessary). - Add 10 ml 0.25% trypsin in PBS-A per embryo; add a sterile teflon or silicone rubber covered magnetic stirrer to the flask and stir at low speed (1 turn/s) for 10 min. - Allow large clumps to settle and remove the turbid supernatant into a centrifuge bottle containing 20 ml calf serum. Add a fresh volume of trypsin to the tissue. -

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Repeat the latter two steps once or twice. If only a few embryos are being trypsinised then the procedure may be carried out by gently shaking the mince and trypsin in a stoppered bottle in a water bath at 37°C for 0.5-1 h. Centrifuge the cells at lOOg for 5 min and discard the supernatant. Resuspend the cells in 20 ml warm growth medium, e.g. medium 199 containing tryptose phosphate (2%) and calf serum (2%). Allow the large clumps to settle and transfer cell suspension to a universal container. Dilute a sample from the cell suspension 1/10 in PBS-A containing 1%acetic acid and count. In a good preparation, 10' cells will be obtained from each embryo. Seed 8 X lo6 cells into 90 mm Petri dishes. Seed 20-30 X lo6 cells into Roux bottles. Only about half the seeded embryo cells attach and grow. If the cell concentration is lowered, the fraction which attaches decreases particularly at seedings of less than 1 X 106/90 mm dish (lo5cells/ml) Incubate cultures at 37°C and change the medium on the following day. Subculture between the third and seventh day after explantation by dividing 1 to 4 ensuring not less than lo5 cells/50 mm dish, and 5 X 105/90 mm dish.

Chick embryo cells are difficult to sustain for more than four or five subcultures without undergoing a marked reduction in growth rate.

6.6. Chick embryo liver cells Remove the embryo as described above. Using forceps and larger scissors remove liver aseptically and immerse in PBS-A in a weighed Petri dish. Repeat until approximately 0.8-1.0 g of tissue has been obtained. - Cut into small pieces using fine scissors and forceps. -

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Discard the medium and replace with 10 ml 0.25% trypsin in PBS-A at 37°C. Incubate for 15 min at 37°C in a humidified incubator (in an incubator containing a tray of water so as to maintain high humidity and thereby prevent evaporation of water from growth media etc.). Discard the trypsin solution and wash the tissue twice with fresh PBS-A at room temperature in a bijou bottle. Immediately dissociate the tissue in a small volume of PBS-A by repeated aspiration. Do this by drawing the suspension into a Pasteur pipette or syringe and expelling it 10 times. Inject the concentrated cell suspension into 10 ml EC,, (Appendices 1 and 6) and repeat the aspiration a further 10 times. Allow undissociated tissue fragments to sediment and remove them. Count a sample of the cell suspension, and adjust the volume of the suspension (with EC,, - Appendices 6 and 1 - at 37"C)to give a cell density of around 3 X lo6 cells/ml. Transfer 5 ml aliquots of the cell suspension to 5 cm Petri dishes and incubate at 37°C in a humidified CO, incubator. After 1 and 3 days remove 2.5 ml medium from each dish and replace with 2.5 ml fresh EC,, (Appendices 6 and 1).

During the first week of growth these primary liver cell monolayers show differentiative changes typical of chick liver at the time of hatching, e.g. chick liver UDP-glycuronyltransferase increases in specific activity up to 10-fold within a day of hatching and similar changes are reproduced in the monolayer (Skea and Nemeth, 1969).

6.7. Rat hepatocytes A perfusion apparatus can be constructed and used to perfuse a rat liver with a solution of 0.058 collagenase in PBS-A (Seglen, 1976). Following treatment the released hepatocytes can be plated out onto collagen surfaces in the presence of 10% bovine serum, insulin and glucagon (10 pg/ml) and hydrocortisone (1 pM). Attachment occurs within 3 h but no cell division occurs though liver specific enzymes

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continue to be produced for several weeks if the serum is replaced by a mixture of fatty acids (Dich et al., 1988). In the presence of EGF, DNA synthesis is induced within 24 h (Hayashi and Carr, 1984). An alternative procedure (Takaoka et al., 1975) involves digestion of minced liver tissue with dispase (0 4.1.4) at 2 I.U./ml medium containing 10% foetal bovine serum (FBS). As dispase is non-toxic, treatment can continue until the bulk of the tissue is dissociated. After filtration the cells are sedimented and seeded at 500-5000 cells per ml medium containing 10% FBS. Erythrocytes disappear from the culture after 1 day and a monolayer of dividing epithelial cells is present by 10-14 days. Fibroblasts are discouraged by a) removing aggregates from the cell suspension prior to seeding, b) using a reciprocal shaker rather than a magnetic stirrer for disaggregation and c) incubating for several weeks in arginine deficient medium during which period any fibroblasts disappear.

6.8. Primary kidney cells This method is applicable to kidneys of various mammals when allowance is made for scale. Remove the kidneys aseptically. In order to do this it will be necessary to shave the animal and then swab the skin with 70% ethanol before making the incision. The dissecting instruments should be dipped into ethanol and flamed before use. - Rinse the kidneys in sterile BSS and transfer to a sterile Petri dish for dissection. - Remove the capsule. This may be done by holding the kidney on edge and slicing it into two halves with a scalpel. The capsule is not severed at the bottom and the two kidney halves may be peeled away from it. - Using fine scissors separate the cortex from the medulla and discard the latter. - Chop up the cortex into a mush using a razor blade held in artery forceps. -

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Flood the chopped tissue in BSS (Appendix 1) and remove the BSS with a Pasteur pipette. Transfer the tissue to a flask with an equal volume of 0.25% trypsin in PBS-A at 37°C (Appendix 1). The flask may be a special trypsinisation flask obtainable from Bellco Glass Inc., NJ, or may be a Virtis or MSE homogenisation flask. Stir mechanically at 37°C at maximum speed possible without causing frothing. After 0.5-1 h allow the tissue to settle and decant the cell suspension. This should be stored on ice until the next step. Further trypsinisation cycles (2 or 3) are carried out until all the tissue is disaggregated. Sediment the cells from the pooled suspension by centrifugation at room temperature at very low speed (50-100 g for 20-30 min). Remove the supernatant fluid using a sterile Pasteur pipette attached through an aspirator bottle to a water pump. Resuspend the cells in prewarmed growth medium and filter through muslin or cheesecloth (filter funnels with muslin held in place with autoclave tape can be wrapped in aluminium foil or Kraft paper and sterilised by autoclaving). A yield of about lo8cells/g of cortical tissue can be obtained and these should be diluted to 3 X lo5 cell/ml and distributed into flasks for growth. Melnick’s medium A (Melnick, 1955) was designed for growth of primary monkey kidney cells but other media have been devised for selective growth of cells of different origins (8 5.4).

A variant of the original procedure was devised by Lieberman and Ove (1962). This led to the isolation of rabbit kidney tubule fragments rather than individual cells (Fig. 6.3) and a special serumless medium was devised for their cultivation. Within 2 days the cells grew out from the tubule fragments to form a cell monolayer. This system proved very useful in investigating the biochemical changes accompanying the changeover from a non-growing population of kidney cortex cells in vivo to a dividing in vitro population (Lieberman et al., 1963; Adams et al., 1966; Lee et al., 1970).

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Fig. 6.3. Rabbit kidney tubule fragments. In the medium devised by Lieberman and Ove (1962) these tubule fragments immediately attach to the glass surface and growth is induced so that within 2 days a monolayer of cells is formed. (Reproduced from Lieberman and Ove, 1962, with kind permission of the authors and publishers.)

Taub and Sat0 (1980) perform a two stage disaggregation. They first digest the mince with collagenase (1 mg/ml) in the presence of soybean trypsin inhibitor (1 mg/ml) and isolate nephron fragments which are then dissociated with 0.1% trypsin, 0.356 EDTA. They developed a serum free medium which did not support the growth of fibroblasts but only the growth of the epithelial cells. This growth medium was a 50 : 50 mixture of DMEM and Ham’s F12 containing 10 mM Hepes buffer and sodium bicarbonate at 1.1 mg/ml and supplemented with Na,SeO,. 5 H 2 0 (10 nM), insulin (5 pg/ml), PGE (25 ng/ml), T3 (5 pM), hydrocortisone (50 nM) and transferrin (5 P g / W Later work (Chung et al., 1982) showed different additives supported growth of cells from different regions of the nephron.

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6.9. Endothelial Cells These can be isolated from veins (including human umbilical cord veins) by a modification of the method of Jaffe et al. (1973) (Astaldi, 1983). The veins should be cannulated and washed in PBS-A containing glucose (11.1 mM) to remove traces of blood and then filled with a solution of trypsin-versene (Q 4.1.5) or dispase (Q 4.1.4). After 15 min at 37°C the contents are collected and the vein rinsed again with PBS-A/glucose. The pooled samples are mixed with growth medium containing 30% autologous serum and the cells sedimented for 5 min at 200 g. The cells are then resuspended in growth medium at lo5 cells per ml and 10 ml inoculated into a 75 cm2 flask precoated with fibronectin. After 5-6 days a monolayer of cells is formed. The medium needs replacement every other day.

6.10. Mammary epithelial cell cultures (R.E.Leake, personal communication) 1) Dissect biopsy samples to remove fat and necrotic regions in medium containing equal amounts of Ham’s F10 and DMEM supplemented with antibiotics and antimycotics. 2) Prepare a fine mince and wash twice with dissection medium by decantation. 3) Resuspend in medium supplemented with FBS (lo%), insulin (5pg/ml) and collagenase (200 pg/ml) and incubate for 1-5 days at 37°C. 4) From time to time gently pipette the suspension; allow clumps to settle and remove the supernatant. The clumps can be redigested with fresh enzyme. 5) Filter the cell suspension through a 0.2 mm nylon mesh and sediment the cells (200 g , 5 min). 6) Suspend in growth medium and plate out onto feeder layers (6 8.1.4).

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6.11. Colonic epithelial cells (Vidrich et al., 1988) Cells can be obtained from biopsy samples after extensive washing in PBS containing antibiotics and antimycotics. 1 cm squares of tissue can be incubated with dispase as described for endothelial cultures. (8 6.9) or with PBS-A supplemented with 1 mM EDTA. Cells are plated out onto feeder layers (0 8.1.4).

6.12. Rat or chick skeletal muscle cells (1-2-day-old rats must be used as older rats yield only fibroblasts.) Remove the thigh muscle and trypsinise in a manner similar to rabbit or monkey kidney. The final cell suspension should be filtered through a double layer of sterile lens cleaning tissue fixed in a Swinnex Millipore filter holder (see 0 2.4) mounted on a 20 ml syringe (Yaffe, 1973). For differentiation the culture vessel must be coated with collagen or gelatin: dissolve gelatin to 0.01% in hot distilled water and autoclave. Put 3 ml into a 6 cm dish and leave in the cold for 2 h. Remove the gelatin solution and add the cell suspension in a mixture of medium 199 (Morgan et al., 1950) and Dulbecco’s Modified Eagle’s medium (1 : 4) with 10% horse serum and 1%chick embryo extract (Appendix 1). Cells plated at 2-3.5 X lo6 cells/6 cm plate attach and multiply for 2 days but at about 50 h the cells enter a period of cell fusion to form a network of multinucleate fibres and cross-striations become obvious (8 15.4).

6.13. Mouse macrophage cultures (see Nelson, 1976) Kill the mouse (6-week-old mice are best) by cervical dislocation and pin to a board. - Spray with 70% ethanol and remove the skin from the abdominal wall. Do not puncture the abdominal wall. -

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Inject 2.5 ml growth medium (Eagle’s MEM plus 10% calf serum and heparin at 10 U/ml), together with some air into the peritoneum. Do not puncture the gut. The abdomen balloons up sealing the site of injection. - Massage the abdominal wall for 1 min to release macrophages into suspension. - Using a syringe fitted with a 21 g X 1; inch needle aspirate the fluid. Insert the needle-eyelet inwards, into the flank and pull sideways to prevent blocking. -

- Count the cells and distribute into dishes (lo6 cells/5 cm dish). Macrophages will attach within 30 min and contaminating lymphocytes and fibroblasts may be removed. Peritoneal macrophages do not normally grow in vitro unless conditioned medium is used. A macrophage growth factor is considered in 0 2.5.

6.14. Ascites cells It is common to passage hybridoma cells through isogenic animals in the form of ascites tumours. The animals may be primed with an injection of pristane (tetramethyl pentadecane, Sigma Chemicals Company - Appendix 3), but this is not always necessary. 106-107cells should be injected intraperitoneally and they should be harvested 7-10 days later before the animal gets too fat. - Harvest the cells aseptically using a sterile syringe and needle and sediment them at 800g for 5 min and remove the supernatant (from which monoclonal antibodies may be prepared). - Resuspend the cells in growth medium and transfer to a 75 cm2 flask containing about 2 X lo7 ascites cells in 10 ml medium. - Gas with 5% CO, in air. - The ascites cells may be contaminated with red blood cells (which are rapidly diluted out) and fibroblasts. - Add further medium (10 ml) after a day and transfer non-attached cells to fresh flasks after 2 and 4 days to remove fibroblasts. -

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6.15. Dipteran cell culture (Doljini, 1971) Horikawa and Fox (1964) isolated single cells from Drosophila embryos and were able to propagate these with the diploid chromosome number for years. - Remove the chorion from blastodermal embryos by treatment with 3% NaClO for 2 min. - Sterilise by treating for 15 min with 0.05% HgCl, in 70% ethanol. - Dissociate the developing eggs by gently homogenising in a glass homogeniser and filter off the vitelline membranes using a 170-220 pm filter. - Wash the cells in growth medium (pH 6.5) supplemented with 10%newborn calf serum. Several cell lines have been isolated (Echalier and Ohanessian, 1969) and one of these has the following phase times (Dolfini et al., 1970) : G1 S G2 T

1.8 h 10.0 h 7.2 h 18.8 h

Cell lines of Aedes aegvpti (Grace, 1966) and A . albopictus (Singh, 1967) have been isolated as follows: Sterilise eggs as above and allow to hatch under aseptic conditions. - Chop freshly hatched embryos and treat with 0.25%trypsin for 10 min at 37°C dispersing by pipetting. - Wash the cells and incubate at 28OC in the medium of Mitsuhashi and Maramorosch (1964) supplemented with 10-20% FBS at p H 7.0-7.4 to which non-essential amino acids and glutamine are added. -

In this medium cells of A . albopictus will double every 10-11 h and have the following cell cycle phase times calculated using the graphical analysis method (Fig 10.12).

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G1 S G2 M

115

3.0 h 4.9 h 1.5 h 1.1 h

These figures are quite similar to those found in vivo for brain cells of A . Aegypti (Marchi and Rae, 1978).

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CHAPTER 7

Techniques 7.I . Cell cloning and plating efficiency Due to inadequacies in the early growth media the first successful cloning of a somatic cell was not acheved until 1948 (Sandford et al., 1948). The problems that arise with cloning are a result of the attempts to grow cells at very high dilutions. In this case the medium must provide nutrients whch may be the products of cell metabolism, but which escape from the cells before they can be utilised in subsequent biosynthetic reactions. Cells growing in culture constantly lose nutrients to the medium even while they remove nutrients from the medium for growth. For cells growing at concentrations of lo5106/ml the medium quickly becomes conditioned. This means that the medium now contains a number of components released into it from the growing cells. These components may be simple amino acids, e.g. glycine, not normally present in simple media (5 2.3.1) or more complex growth factors (0 5.8). However, when the cell concentration is reduced to 1 or 2 cells/ml the outflow of, for instance, glycine cannot be balanced by an equivalent uptake and cells die of starvation. The requirements of cells growing at low density include pyruvate and non-essential amino acids and also carbon dioxide (Eagle, 1971). It is therfore important either to clone cells using a bicarbonate/CO, buffer or, if organic buffers such as Hepes are used, they must be supplemented with bicarbonate and CO, (e.g. 20-25 mM Hepes plus 8 mM bicarbonate and 2% CO, in air gas phase). Two approaches have been used to circumvent this problem. Cells are either cloned in a restricted microenvironment, e.g. a capillary tube in which they can rapidly condition the medium, or they are cloned in the presence of a large number of killed cells, i.e. a feeder layer irradiated with X-rays (Puck et al., 1956; Sandford et al., 117

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1961). Ham's medium F12 has been designed specifically for cloning cells (Ham, 1965) and should be supplemented with 10-308 foetal bovine serum. Great care must be taken to prevent the medium becoming too alkaline by loss of CO,. These methods are, however, largely unnecessary when it is desired to clone continuous cell lines. Thus a dilute suspension of HeLa cells (containing about 100 cells in 5 ml) will grow up to form colonies, each derived from a single cell. It is important to prevent the movement of these cells (and hence mixing of clones) and sometimes they are overlayered with soft agar. The clones may be isolated either by surrounding each colony with a stainless steel cylinder and removing the cells by trypsin action or by initially seeding the cells into a multiwell plate and selecting those wells in which single colonies become established. If care is taken, single cells can be selected from a suspension and transferred to individual wells in a multiwell plate. For measuring plating efficiency a similar procedure is adopted and the colonies are fixed and stained (with Giemsa) and their number compared with the initial input. The plating efficiency of primary cells is low, but plating efficiencies of up to 100% can be achieved with cell lines or cell strains. 7.1.1. Measurement of plating efficiency

Wash the cell monolayer in BSS to remove serum. Release cells with 0.25% trypsin in PBS or BSS (the divalent ions improve plating efficiency) and disperse by gently pipetting up and down the minimum number of times. Trypsinisation at 4°C for 2-10 min has been recommended especially for cloning cells in low serum. - Add an equal volume of complete medium. Medium F12 supplemented by up to 30% foetal calf serum has been recommended (Ham, 1965). The medium should be preincubated with 5% CO, to prevent undue alkalinity and should not be above 30°C, otherwise the cells will attach to the walls of the vessel (Ham and Puck, 1967). -

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Dilute so that 5 ml contains between 50 and 250 cells and quickly inoculate into a 5 or 6 cm dish (see cloning, method 1 below). - Incubate for 8-15 days at 37°C in a humidified CO, incubator (0 3.1.1). If the cells migrate quickly intermixing of colonies may occur. To prevent this, medium containing 0.17% agar may be used (9 7.1.3). - Fix the resulting clones in 25% formalin and stain with Giesma (Appendix 2) to estimate plating efficiency. -

7.1.2. Four simple cloning methods

Method 1 Cells should be dispersed as described in 0 7.1.1, but the culture vessel should contain many small fragments (0.05 cm2) of coverslips to whtch the cells will attach and grow. Using sterile forceps remove coverslips fragments on which single cells have attached (check this microscopically) and transfer them to separate wells in a tissue culture tray (0 3.2). -

Add 0.3 ml complete medium to each well and continue the incubation until the clone covers the surface of the well. The cloned cells may now be harvested by trypsinisation (0 4.2) and transferred to appropriate vessels.

Method 2 - Clones may be isolated from 5 cm dishes using stainless steel cloning cylinders (about 6 mm in diameter and 12 mm high (Dow Corning; Appendix 3). These, and some silicone grease should be sterilised by autoclaving. - Mark the position of a well isolated colony of cells using a grease pencil or magic marker on the undersurface of the dish. - Remove the medium and rinse with BSS (Appendix 1). - Using sterile forceps touch one end of a cloning cylinder into the silicone grease and place it over the colony to be isolated. - Using a syringe and a 20 gauge needle add a drop of 0.05% trypsin in BSS to the cylinder to loosen the cells which should then be withdrawn back into the syringe and transferred to a fresh Petri dish.

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Method 3 - The dilute cell suspension may be plated out on special Petri dishes whose growth surface is made of a membrane (Petriperm: Appendix 3). This membrane allows ready diffusion of gases in and out of the dish from below and is also readily cut into pieces. In this way pieces containing single colonies may be cut out and transferred to the wells in a tissue culture tray (cf. broken coverslips). Method 4 - Fill each well of a 96 well flat bottomed microtitre tray (Linbro Chemical Co., Inc., or Titertek Inc.; Appendix 3) with 50 pl growth medium. - Prepare a cell suspension containing about 4000 cells per ml taking care that cells are not clumped or even sticking together in pairs, and pipette 50 p1 into the 8 wells in the first row of the tray. - Mix and remove 50 pl to the wells in the second row leaving about 100 cells in each well of the first row. - Continue to the end of the tray to give a set of 2- fold, serial dilutions of the original suspension. An %place dispenser is invaluable for this operation. Several of the wells in row 7 or 8 should contain a single cell and this can be checked microscopically. - Incubate the tray at 37°C in a CO, incubator until the clones develop (7-10 days) when they can be removed by gentle trypsinisation to the wells of a 24-well plate or to 25 cm2 flasks. (In each column, the last well to contain growing cells probably contains a clone.) An alternative method involves using a microcapillary to select single cells which are transferred to individual wells in a microtitre tray. 7.1.3. Cloning under agar

Under certain conditions cells will form colonies when not attached to a glass or plastic substratum but when suspended on soft agar. Macpherson (1973b) points out factors which promote the growth of

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121

cells in soft agar. These involve transformation with oncogenic viruses, infection with mycoplasma, increased levels of serum and addition of feeder cells or DEAE dextran to neutralise the acidic groups present in agar. A 1.25% solution of Difco Bacto-agar is made up in hot distilled water and sterilised by autoclaving. The agar sets on cooling and must be melted in a boiling water bath and cooled to 44°C before use. - Add 20 ml calf or foetal calf serum and 20 ml tryptose phosphate broth (Appendix 1) to 80 ml double strength medium (e.g. Glasgow MEM; Appendix 1) and warm the mixture to 44°C. - Add some of the melted agar to the above supplemented medium and mix gently to yield 0.5% agar medium. - Pipette 7 ml 0.5% agar medium into 5 cm Petri dishes and allow to set at room temperature. Use within 1 h. - Mix 1 volume of cell suspension with 2 volumes of 0.5% agar medium at 44OC and add 1.5 ml as an upper layer onto the agar base layer prepared above. Up to l o 3 colony-forming cells should be present per dish. - Incubate in a humidified CO, incubator for 7-10 days during which time colonies of 0.1-0.2 mm diameter form. - Colonies may be removed from the agar into a tube containing 1 ml medium using a finely drawn pipette. - Pipette the suspension up and down to break up the agar and release the cells into suspension. - Transfer the cells to 5 cm dishes and add 5 ml agar-free medium. Incubate in a humidified CO, incubator (0 3.1.1). -

7.1.4. Cloning with a feeder layer

Confluent cultures containing (2 x lo6 cells/90 mm dish are irradiated with 4000-6000 rads of y-radiation from a cobalt source. Irradiation should last for less than 1 min and a variety of cell lines (e.g. HeLa, BHK21/C13 or 3T3) are suitable (Puck et al., 1956). These cells may be used directly or may be trypsinised into smaller vessels. They may appear healthy for up to 4 weeks but do not divide.

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An alternative method of producing a feeder layer is to incubate the monolayer with mitomycin C (10-6M) for 16 h which causes crosslinking of the DNA (Iyer and Szybalski, 1964). The monolayer is then washed three times with BSS to remove the mitomycin C and may be used directly or after subculture into smaller vessels. For cloning 6 cm dishes containing about 2 X lo5 killed cells (i.e. irradiated or treated with mitomycin C ) are used. These form the layer of feeder cells. Replace the medium in the dish of feeder cells with a suspension of the cells to be cloned. As few as 10 cells may be present but about 1000 is better. These cells settle into open spaces between the feeder cells and begin to grow and form colonies which may be isolated using cloning cylinders (0 7.1.2). Spleen lymphocytes (0 13.6.1) can also be used as feeder cells. These cells do not normally divide, nor do they attach to the surface of the dish and they disappear following 6-10 days in culture.

7.2. Cell counting procedures There are two methods of estimating the number of cells in a suspension. Using a haemocytometer the number of cells in a given volume is counted by direct microscopic examination. Using electronic counters, e.g. the Coulter counter, the cells in a given volume of suspension are drawn through an orifice and registered electronically. 7.2.1. Haemocytometer

Place the precision ground coverslip on the haemocytometer slide (Fig. 7.1) so as to cover the two ruled areas and press down gently until Newton’s rings are visible. T h s leads to formation of a chamber of precise dimensions as the edges of the slide are raised exactly 0.1 mm above the ruled area. Each ruled area consiste of 9 large squares, each of 1 X 1 mm, i.e. the volume above each large square is 1 x 1 x 0.1 = 0.1 mm3. (The 4 corner squares are subdivided into 16 smaller squares, the centre square into 25 smaller squares and the

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123

Fig. 7.1. Use of the haemocytometer. The diagram on the left shows a modified Neubauer haemocytometer and on the right is a photomicrograph of BHK21C13 cells ready for counting.

remaining squares into 20 smaller squares, but t h s is unimportanr. when counting cells.) A drop of cell suspension (containing between lo5 and lo6 cells/ml) is placed at the two edges of the coverslip so that the suspension flows into the chambers by capillary action (Fig. 7.1). Do not flood the chamber. The slide is viewed under a low power objective of a microscope. The cells in four large squares in the comers of each of the two ruled areas are counted. Count cells touching the right and top lines but not those touching the left and bottom lines. The volume of a large square is 0.1 mm3 and the average cell count should therefore be multiplied by lo4 to give the number of cells per ml. 7.2.2. Electronic cell counter

The principle of t h s method is that as a cell passes through an orifice it interrupts an electric current and this interruption is picked

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Fig. 7.2. Diagrammatic representation of an electronic cell counter. When the upper tap is open the vacuum pump draws the mercury into the position shown in black (lower position). On closing the tap the mercury slowly returns to its equilibrium (upper) position and while so doing it draws cell suspension through the small orifice. As a cell passes through the orifice it interrupts the current flowing between the two electrodes and each cell is registered on an oscilloscope and is counted. The count is recorded as the mercury sweeps out the volume between the switches (usually 0.5 ml).

up as an impulse which is translated into a visual signal on an oscilloscope and recorded on a counter (Fig. 7.2). The size of the visual impulse is proportional to the volume of the cell and hence electronic ‘gates’ can be set to count cells of given sizes. The simpler machines (e.g. Coulter counter model D industrial) have only a lower threshold to the gate which ensures that particles of dust are not counted. The more complex machines (e.g. Coulter counter model ZM) have variable upper and lower thresholds and will measure cell number with automatic coincidence correction if required and average cell diameter or volume. When coupled with the Channelizer 256

CH. 7.

TECWIQUES

Cell

125

volume p3

Fig. 7.3. Electronic cell volume plotter. Mouse L929 cells were harvested from stationary phase cultures at zero time and subcultured into medium to which thymidine (5 mM) was added at 8 h. At 24 h the thymidine was removed. At various times the cells were harvested by trypsinisation and their volumes measured using a Coulter counter model B fitted with a model J plotter. -, 0 h (Go cells); _ _ _ , 28 h (late S-phase cells); . . . . . ., 32 h (Gl-phase cells). (Reproduced from Adams, 1969b. with the publisher's permission.) These models have now been replaced with the Coulter counter model ZM with the C256 Channelyzer.

a plot of distribution of cell population with size can be obtained similar to that shown in Fig. 7.3. The orifice through which the cells pass can vary in size but for animal cells the most suitable is 100 pm in diameter and 75 pm long and is manufactured from a ruby. The orifice tube is therefore fragile and expensive. Prepare a counting saline solution (NaC1,0.7%; citric acid, 1.05%; mercuric chloride, 0.1%)and to 24.5 ml in a small beaker add 0.5 ml of cell suspension (approx. lo6 cells/ml); alternatively 0.7 ml suspension may be added to 16.8 ml counting saline in a universal bottle. Mix the saline suspension well and set it on the counting table with the orifice tube and outer electrode immersed. Open the

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upper stopcock which connects the mercury manometer to a vacuum produced in a bottle by a small pump. This draws the mercury down the manometer. After a few seconds the stopcock is closed and the mercury in the manometer returns to its original position. As it does so it draws cell suspension through the orifice. At the same time the mercury switches on the cell counter as it passes a wire electrode (a on Fig. 7.2) and switches it off again as it passes a second wire electrode (b on Fig. 7.2). Between the two electrodes the mercury sweeps out exactly 0.5 ml and hence draws this volume of saline cell suspension through the orifice. In the standard procedure 0.5 ml of a 1/50 dilution (i.e. 0.01 ml) of the original suspension is counted but this may be varied depending on the concentration of the original suspension.

7.2.3. Comparison of the methods The disadvantage of using the haemocytometer is that it becomes tedious for large numbers of samples. Moreover, there is an error of about 10% in estimations (Sandford et al., 1951). Some of the errors arise during dilution of the cell suspension which should be done with care. An advantage is that the apparatus required is quite cheap (Neubauer counting chambers can be obtained from Gelman Hawksley) and very small numbers of cells are required. It is thus easy to estimate the number of cells in a small dish by trypsinising them into a volume of 1 ml or even in a microtitre plate well in 50-100 p1. Because the cells are viewed directly, this method enables a judgement to be made about the quality of the cell suspension, i.e. clumped or broken cells present. By diluting the cell suspension with a solution of a vital dye, e.g. trypan blue at a final concentration of 0.2 g/1 PBS-A (Appendix l), and counting only the unstained cells a measure of the viable cell count is obtained. (N.B. Trypan blue is toxic and should not be allowed to come in contact with the skin.) The electronic cell counter is, however, more reproducible. As many thousand cells are counted, machine sampling errors are very small and errors of less than 5% are easily achieved. The main source

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127

of error is in the initial sampling of the cell suspension which must be done with care. The number of cells in a small dish may also be estimated using an electronic counter but in this case almost all of the cell suspension is used in the estimation. If two or more cells pass through the orifice simultaneously they will be counted as one and corrections must be applied with cell concentrations above 104/0.5 ml. The coincidence error at this concentration is 3% but it rises to 22% at 105/0.5 ml. One big advantage is the visual display on the oscilloscope of the range of cell size present in the suspension. On the one hand, thls indicates if clumps of cells are present, and on the other it gives a measure of the distribution of cells around the cell cycle (see Chapter 10). In addition, the use of gates or windows in the Coulter counter model ZM enables cell sizes to be accurately determined after the machine has been calibrated with pollen grains of known size. Thus ragweed pollen has a mean volume of 3800 pm3. When coupled to a ‘Channelyzer’ a histogram of the cell size distribution is automatically recorded (Fig. 7.3). The Coulter model D Industrial counter costs &4250 and the model ZM costs g.8500. The Coulter Channelyzer model C256 costs E6500 (1989 prices).

7.3. Storage of cells The long-term storage of cells at low temperature is now routine. Methods awaited the observation that glycerol exerts a marked protective effect on the cells during the freezing process. During freezing (particularly during rapid freezing) ice crystals form within cells which adversely affect lipoproteins and cause the splitting of cell membranes. If cells are cooled quickly the concentrations of salts in the extracellular medium increases producing osmotic damage. Water is lost from cells which therefore shrink. If t h s is allowed to occur slowly and to completion then it is a dehydrated cell which is preserved and no intracellular ice crystals are present (Mazur, 1977). Substances which protect cells must be non-toxic, of low molecular weight and high solubility and must be readily able to

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permeate living cells (Lovelock and Bishop, 1959). Glycerol is still the most preferred protective agent but some cells (particularly bovine red blood cells) are only permeated slowly. Dimethylsulphoxide (DMSO) is commonly used as an alternative to glycerol. In general, slow freezing and rapid thawing is recommended for maximum survival. The freezing should be at the rate of l"C/min which is generally achieved by placing the cells in a box of expanded polystyrene (about 1.5 cm wall thickness) in a -70°C freezer for at least 2 h before transfer to liquid nitrogen. Freezing at l"C/min allows sufficient time at -20°C to -30°C for cell shrinkage. At - 196°C cells may be stored for many years without substantial loss of viability. Some cell lines, e.g. BHK C13 may be stored for many months at -70°C and this is very convenient for those cells in constant use in a laboratory. Programmable freezers are available that, when coupled to a liquid nitrogen supply, ensure that the rate of cooling over the critical temperature range is very carefully controlled. Initially the temperature is programmed to fall at 1°C per min. The major problem is that at the point of freezing the release of latent heat can have the effect of subjecting microenvironments to repeated freezethaw cycles. In the Cryoson machine (Appendix 3 and Fig. 7.4) this is countered by injecting a pulse of liquid nitrogen at this point to neutralise any temperature increase and allow rapid freezing. Subsequently the temperature is lowered to -120°C at 4°C per minute. Vials are then transferred to a liquid nitrogen freezer. In order to use a programmable freezer it is essential to have a supply of piped liquid nitrogen from a pressurised cylinder and this is also very convenient' for topping up liquid nitrogen freezers. Containers of various sizes (e.g. 200 1) can be obtained from, for example, British Oxygen Company, Planer Biomed, Taylor-Wharton, Statebourue Cryogenics or Thermolyne (Appendix 3) and these are replenished as necessary on a weekly or daily basis by the supplier. The larger ones should be situated outside the laboratory to allow access for delivery and space for vapour to disperse and an insulated pipe can be connected to the freezer (Fig. 7.4). Automatic systems are available but have been known to malfunction leading either to warming of cell stocks or to the overflow of liquid nitrogen from the storage vessel.

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Complete 96 well microtitre plates and their contents can also be frozen in a programmable freezer. The medium in the wells should be replaced with freezing medium (0 7.3.1) and the whole plate cooled on ice and taped shut and wrapped in plastic film prior to freezing. 7.3.I . Freezing procedure

Grow the cells for 2-3 days in 75 cm2 flasks until a sub-confluent monolayer is formed. it is advisable to change the medium on the cells 24 h before they are due to be harvested. - Harvest the cells from each flask into a separate universal container. Count the cells. Take a bacteria check from each bottle and keep lo6 cells for a mycoplasma check (Chapter 9). - Centrifuge at 250 g for 5 min to pellet the cells. Meanwhile prepare storage medium which is normal growth medium containing 10%glycerol (or DMSO) with an increased concentration of serum. Hybridomas are best stored in 90% FBS plus 10%DMSO but a more common medium is:

-

Eagle’s MEM 65 ml 25 ml Calf serum Glycerol 10 ml The glycerol should be sterilised by autoclaving at 15 lb pressure for 15 min. - Resuspend the cells in storage medium to contain 5-6 X l o 6 cells/ml. If DMSO is used as the cryopreservative this should be done at 0°C . As an alternative to pelleting the vessels the cell suspension may be diluted with glycerol (or DMSO) containing medium to give a concentration of 10%. - Using a Pasteur pipette, dispense approximately 1.5 ml aliquots into sterile ampoules (biofreeze vials, Fig. 7.5). These may be blue spot or yellow band, easy snap glass ampoules (Epsom Glass Industries Ltd.) but plastic vials (Nunc, Costar or Sterilin) are now more common. The round bottom vials are difficult to work with and the standard flat bottom vials are very fiddly. Bibby Science Products (Appendix 3) supply a cryogenic work station -

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Fig. 7.4(a). A vapour phase liquid nitrogen freezer showing one of the sections partly removed. There are six sections each with five drawers each of which holds 55 vials. In the background is a Cryoson programmable freezer showing the temperature probe and a rack of vials. The liquid nitrogen is piped to the programmable freezer from a 200-1 tank kept outside the building (b).

CH. 7.

TECHNIQUES

Fig. 7.4 (continued)

131

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which holds vials firmly (in ice if required) so that they are easy to open with one hand without fear of their toppling over. - Seal ampoules carefully (glass ampoules are sealed using a bunsen flame; plastic ampoules are sealed using the screw cap which is much simpler). - Test for improperly sealed glass ampoules by immersing in ethanolic methylene blue solution. The dye will penetrate those ampoules which are improperly sealed. - Rinse ampoules in 70% ethanol, dry and label. - Freeze the samples as described above. 7.3.2. Storage procedure When the samples are frozen (at - 70°C or - 12OOC) they should be transferred to a liquid nitrogen freezer. These are of two types. In the older version the vials are actually immersed in liquid nitrogen but in the newer models the samples are simply held in the vapour above a sea of liquid nitrogen. For the liquid phase freezer, ampoules are transferred to canes (Fig. 7.5) or cryoboxes and immersed CAREFULLY into the liquid nitrogen vat. Gloves and goggles or visors must be worn to protect yourself from splashes of liquid nitrogen. Canes have the problem that vials can be lost from them or are difficult to remove when required, and the locator system of cryoboxes (Thermolyne; Appendix 3) has advantages. For the vapour phase freezer, ampoules are placed in storage racks which are lowered into the freezer (Fig. 7.4). The liquid phase freezers are more difficult to use but because of their narrow neck they are less likely to lose their liquid nitrogen than are the wide necked vapour phase models. In either case it is imperative to top up with liquid nitrogen to the required level twice a week. It is a good idea to keep valuable cells in two different freezers. Pathogenic samples must be stored in vapour phase to avoid spread of contamination on explosion. Good records are important, otherwise freezers become filled with numerous vials of unknown provenance; and the use of cells for commercial purposes requires a complete history of each vial.

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Fig. 7.5. Materials used for freezing cells. On the right are (top to bottom) a plastic ampoule and a 1 ml and a 2 d glass ampoule. When sealed and cooled to - 70°C these may be snapped into canes (middle) which are put into the container on the left for immersion in the vat of liquid nitrogen. Alternatively vials may be stored in racks as shown in Fig. 7.4.

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Suitable liquid nitrogen freezers (and canes) are available from Union Carbide, British Oxygen Company etc. (Appendix 3). 7.3.3. Recovery of cells from liquid nitrogen When removing glass or plastic ampules from liquid nitrogen, always observe the following precautions: a) wear a perspex face shield b) wear protective gloves c) leave cane aside for 20-30 sec before removing an ampoule. If any ampoules are liable to explode they will do so soon after being taken from the nitrogen. Prepare a 25 cm2or 75 cm2 flask with 5-10 ml of growth medium. Gas and warm thoroughly to 37°C. - Observing all the above precautions remove an ampoule from the cane. - Thaw the ampoule in a 37°C water bath as rapidly as possible using gentle hand agitation. Immerse the ampoule in 70% alcohol to sterilise outside (remember liquid nitrogen is not sterile). - Score the neck of a glass ampoule with a file or diamond and break the ampoule between the folds of a sterile towel. Unscrew the cap of plastic ampoules. - Transfer the contents of the ampoule to the pre-warmed medium in the flask. This is a critical step, especially for cells stored in a high serum concentration. It is preferable to first take the cell suspension into a Ripette and then draw up 5-10 ml of medium slowly. Only when this is mixed should the diluted suspension be added to the rest of the medium in the flask. Incubate at 37°C (in a CO, incubator, if required). - After 24 h decant the medium from the bottle. Keep and test for bacterial contamination. Replace with 10 ml of fresh warm growth medium. - Incubate at 37°C until cells reach confluence.

-

To recover a frozen microtitre plate it should be transferred to a shallow 37°C water bath and shaken as it thaws. In a laminar flow

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hood 100 pl calf serum should be added to each well and the whole plate centrifuged for 5 min at 800 g. The supernatants are removed and the cells washed and recentrifuged prior to incubation in growth medium (de Leij et al., 1987). 7.3.4. Organisation of stocks of frozen cells

Following prolonged subculture of cell strains and cell lines there is a significant chance that the properties of the cells will change. Mutants may arise which will outgrow and replace the origmal cells. In order to ensure that the cells being used in an experiment remain the same over a period of years, it is therefore essential not to sub-culture them too often but to return to frozen stocks at least every two months. On obtaining a cell strain or cell line which is to be used as experimental material over a period of months, one of the first steps is to grow several flasks of cells for freezing. Then every 2 months a fresh vial should be unfrozen and new cultures set up. As soon as these are available in adequate amounts the old cultures should be discarded. Before the stocks of frozen cells fall too low a new batch of cells should be prepared for freezing. On subculturing cells a note should always be made of the passage number and it should be realised that cells of high passage number may not be identical with the original cells. As a laboratory may require several canes of frozen vials for each cell type being carried, this puts a certain strain on liquid nitrogen storage facilities. This is, however, the recommended procedure and does provide a reliable back-up in cases of contamination. 7.3.5. Cell banks and transport of cells

A number of organisations store frozen cells for sale or distribution and will also store cells for individuals as a back-up for departmental liquid nitrogen freezers. These include the European Collection of Animal Cell Cultures at Porton Down, the American Type Culture Collection Cell Repository at Rockville, MD, and the Human Genetic Mutant Cell Repositary at Camden, N.J. (Appendix 3).

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Many other companies also sell a variety of cell lines, often acting as intermediaries between the main depositories and the customer. To use a local company is often an advantage as they take the responsibility of recovering the cells from the frozen state, and they will supply cells delivered in flasks completely filled with medium. When the cells are received frozen they should be thawed out and subcultured as described in 6 7.3.3. If the cells are delivered at room temperature in a flask of medium, most of the medium should be removed (and saved for later use) and the cells allowed to recover by incubation at 37°C in the remaining medium. Suspension cells will require gentle sedimentation prior to resuspending in about 10%of the transport medium. After 1 or 2 days the cells can be subcultured and, as a precaution, some can be grown in the transport medium. To deposit cells (or tissues) at the repositories a frozen vial or a subconfluent culture in a 25 cm2 flask filled completely with growth medium is required. In the latter case the neck of the flask should be taped and the flask wrapped in pop-film in a polystyrene box or jiffy bag. The package should be kept at room temperature and delivered by the fastest route. Depositories require documentation before they will accept samples and the appropriate paperwork must be completed before the cells are despatched.

7.4. Karyotyping The chromosomes constitution of cells, or cell hybrids, is an important criterion for monitoring the nature of cells in culture. It may help to indicate contamination of the cells of one species with those of another and enables the elucidation of the chromosomal location of various genetic markers. In vitro, however, cells often change their chromosome constitution and most cell lines no longer have the diploid number of chromosomes. Rather they are aneuploid and may have a wide range of chromosomes per cell (see 6 2.2) Although it is easy to see the chromosome mass in metaphase cells, the individual chromosomes cannot be distinguished. In order to analyse the chromosomes present in a cell (karyotype) it is

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necessary to swell the cells in a hypotonic buffer so that, when they are dried onto a slide, the chromosomes settle, spread out over a wide area. It is important, however, that the cells do not burst in suspension, otherwise it becomes impossible to distinguish the cellular origin of individual chromosomes. As even in an exponential culture only a small proportion of cells are in mitosis, it is necessary to increase this number by addition of colcemid or nocodazole whch block cells in mitosis (0 10.2). This also has the effect of separating individual chromosomes by its action on the spindle. 7.4.1. Chromosome preparation

The procedure outlined here (Hsu, 1973) is designed to spread out the chromosomes of one cell without leading to their intermingling with those of adjacent cells. (Alternative procedures involve treating a cell monolayer with hypotonic medium before fixation.) a) Treat exponentially growing cells with colcemid (0.06 pg/ml may be sufficient but some cells require up to 5 pg/ml) or nocodazole (0.04 pg/ml) for 2 h (or up to 6 h for nocodazole). b) Trypsinise monolayers and pellet cells quickly. Make sure mitotic cells are not discarded in the medium prior to trypsinisation. c) Suspend the cells in 5 ml hypotonic medium (one part growth medium and 2 parts water or simply 0.5% KCl) and leave for 10 min. c’) As an alternative cells may be resuspended in complete growth medium (to stop trypsin action) and then washed twice in Hanks BSS before resuspending in a small volume (0.5 ml) Hanks BSS to which 2 ml of water is then added. (For Q banding 75 mM KCl is recommended - see below.) Allow the suspension to stand in this hypotonic salt solution for 8-10 min. d) Sediment the cells and add 4 volumes of fixative (3 parts methanol :1 part glacial acetic acid) without disurbing the pellet. e) After 10-20 min (depending on the size of the pellet) resuspend the cells and recentrifuge. f) Resuspend in fresh fixative and recentrifuge.

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g) Repeat (f) twice more, finally resuspending in a small volume (1

ml or less) fixative. h) Wash a slide in alcohol/HCl to ensure that it is grease free and dry with tissue. Add 3 small drops of cell suspension which should spread over the whole slide surface and blow gently to dry (the moisture in the breath is important). i) Stain with acetic orcein (2% orcein in 45% acetic acid, 3-5 min) or Giemsa (Appendix 2). j) Rinse, air dry and mount. A much simpler, though in general less satisfactory procedure, is to grow cells on coverslips and treat as above with colcemid. Treat the coverslip for 30 min with warm, hypotonic saline (see c’ above) and then fix for 10 min and dry at room temperature. Stain the cells and mount the coverslips, cells downwards. The difficulty with this method is that cells are easily dislodged from the substratum on adding the fixative. For this reason this step should take at least 10 min. 7.4.2. Karyotyping

To prepare a karyotype (the chromosome constitution of a cell) a series of metaphase preparations is photographed and the individual chromosomes cut out and arranged in order of decreasing size (an Idiogram - Fig.7.6). In most cases groups of chromosomes are apparent - e.g. in man there are seven groups - but seldom is it possible to identify individual chromosomes solely on the basis of size and position of the centromere. Karyotyping has advanced dramatically since it was discovered that certain stains stain individual chromosomes in characteristic ways. Caspersson (Caspersson et al., 1970a,b, 1971) showed that quinacrine mustard and quinacrine dihydrochloride (Fig. 7.7) produced a characteristic banding pattern (Q-banding), and later it was found that complementary bands could be formed with Giesma (G-bands) if the chromosomes are treated first with trypsin or mild alkali (Patil et al., 1971; Seabright, 1971; Wang and Federoff, 1972).

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C H . 7.

Fig. 7.6. Chromosomes of BHK21/C13 cells. The chromosomes were cut out from a photograph of a metaphase cell chromosome spread and arranged roughly into groups on the basis of size and by reference to the ratios of the lengths of the long and short arms (Marshall, 1972). The autoradiographic grains are the result of an in situ hybridisation with tritiated poly U (Steffensen, 1977). This was achieved by treating the fixed cell preparation with 0.2 N HC1 (to remove basic proteins) and ribonuclease (100 p g / d 2xSSC) before denaturing the DNA in 0.07 N NaOH for 2 min. The preparation was then washed three times in 70% ethanol and three times in 95% ethanol and dried. 10 p1 (140 ng) of ['Hlpoly U in 3XSSC was applied to the coverslip which was incubated at 2OoC overnight before washing thoroughly in 2xSSC. After a second ribonuclease treatment (20 p g / d for 30 min at 2 0 T ) the coverslip was washed in 2XSCC (three times), 70 and 95% ethanol, air-dried and autoradiographed using Kodak ARlO stripping film. (Reproduced from Shenkin, 1974, with kind permission.)

NH. c H c H [C HJ N ( c H

c

H

3

0

m

c H ,)

CI

Quinocrine or o t e b r l n

Fig. 7.7. Quinacrine or Atebrin.

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7.4.3. Q-banding (Caspersson et al., 1970a; Lin et al., 1971)

Use slides on which cells (swollen in 75 mM KCl) have been fixed as described in 7.4,la-h. - Dip the slides into distilled water for a few seconds. Transfer to staining solution (0.5% w/v quinacrine dihydrochloride in 0.1 M phosphate buffer pH 4.5. The stain may be obtained from G.T. Gurr Ltd.; Appendix 3 - under the name ‘Atebrin’). - After 15 min wash the slides in three changes of distilled water for a total time of 10 min. - Air-dry and mount with a drop of distilled water, sealing the edge. - Examine as soon as possible with a fluorescence microscope (excitation filter No.1 (BG12) and barrier filter No.47). -

The initial Caspersson method uses a staining solution of 5 mg% quinacrine mustard in 0.09 M Na,HPO,, 8.7 mM citric acid pH 7.0. After 20 min the cells are washed three times in the same buffer or in distilled water pH 7.0 and sealed wet for visualisation with a fluorescence microscope. 7.4.4. G-banding (Wang and Federofi 1973; Moorhead et al., 1960)

Treatment of metaphase chromosomes with dilute trypsin solutions brings about a removal or redistribution of non-histone proteins along the chromosomes producing banding patterns observable with the phase contrast or interference microscope (Comings et al., 1973; Stubblefield, 1973). The bands can be intensified by staining with Giemsa when dark bands are apparent corresponding to the bright fluorescent Q-bands. Growing cells are treated with colcemid (0.4 pg/ml) for 3 h at 37°C and if they are growing as a monolayer, they are then harvested by trypsinisation. Pellet the cells (200 g for 5 min) and suspend in 5 ml of hypotonic (0.075 M) KCl. - Leave at room temperature for 8 min and then add 1 drop of cold fixative (methanol : glacial acetic acid, 3 : 1). - Mix gently and pellet the cells at 4°C. -

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Remove the supernatant and add fresh fixative gently, without suspending the cells. After 30 min resuspend the cells, pellet, resuspend in fixative and pellet the cells again. Finally resuspend the cells in 0.25 ml fixative and add a drop to a thoroughly clean (degreased) slide. Air-dry quickly. Treat the fixed preparation with trypsin (0.025%) versene pH7.0 for 10-15 min at 30°C. This causes considerable swelling of the chromosomes. The actual time of treatment should be monitored by phase contrast microscopy - the end point being when the two chromatids fuse together and appear as a unit. Rinse in 70%, 80% and absolute ethanol. Air-dry. Stain for 12 min with Giemsa (pH 7.0; Appendix 2). Wash in distilled water. Air-dry.

Alternatively, the air-dried slide may be transferred to a solution of quinacrine mustard dihydrochloride (50 pg/ml in MacIlvaine’s buffer - 6.5 ml 0.2 M Na,HPO,, 43.6 ml 0.1 M citric acid and H,O to 100 ml pH 7.0). After 20 min the slides are washed three times with MacIlvaine’s buffer and sealed wet for visualisation with a fluorescence microscope. Analysing karyotypes by hand is extremely tedious and the increasing demands of the health service have led to the development of several computer operated systems which automatically locate and analyse metaphase spreads (Zeidler, 1988). A manual of chromosome techniques has recently been published (Verma and Babu, 1989). 7.4.5. Chromosome sorting

Metaphase chromosomes can be sorted by flow cytometry in the same way that cells can be sorted (see fj 10.7.5). Usually separation is based on the DNA content of ethidium bromide or propidium iodide stained chromosomes but Hoechst 33258 (which preferentially stains AT rich segments) and chromomycin A3 (which stains CG rich regions) can also be used (Davies et al., 1981).

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In a few cases (e.g. yeast) small chromosomes can be fractionated by pulsed field gel electrophoresis.

7.5. Cell transfection This is a procedure increasingly used to introduce DNA into cells and various methods are available. I have experience with the calcium phosphate technique which is described below but other techniques are described by Gorman (1985) and Spandidos and Wilkie (1984). For example, Bethesda Research Ltd. supply liposomes whch will mediate the uptake of nucleic acids at high efficiency (Felgner and Ringold, 1989) and with practice and the appropriate apparatus material may be injected directly into cells (Ansorge and Pepperkok, 1988). Electroporation is particularly useful for introducing DNA into plant spheroplasts. 7.5.1. Calcium phosphate method

It is important to use cells which are growing exponentially and the efficiency of transfection is poor in slowly growing cells. Plate out cells at 3 X lo5 per 25 cm2 flask in 5 ml growth medium and incubate overnight. Prepare DNA solutions aseptically in 1 mM Tris HCl/O.l mM EDTA pH 8.0 and prepare a sterile 2 M CaC1, solution. Also prepare a 2 X HBS solution: 280 mM NaCl (1.63%) 50 mM Hepes (1.19%) 1.5 mM Na, HPO, . 2H,O (0.023%) Adjust to pH 7.1 with NaOH and filter sterilize. Mix 835 p1 sterile water with 125 pl 2 M CaCl, and 40 pl DNA This should contain 1-10 pg test DNA and be made up to a constant amount (40 pg) of DNA by including carrier.

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Add the DNA/CaCl, solution to an equal volume of 2 x HBS. This should be done slowly (over 30 sec) with continuous mixing in the order stated. Vortex and allow to stand at room termperature for 30 min until a fine precipitate forms. Method A : suitable for most cells. Add 1 ml DNA precipitate to each flask (do not remove the medium) and incubate overnight. Remove the medium, wash the cells with prewarmed PBS and add 5 ml fresh prewarmed growth medium and return the flasks to the incubator. Method B : required for some cells which are difficult to transfect. Remove the medium from the flask and add 1 ml of the DNA precipitate. Incubate 37°C for 45 min and then add 4 ml growth medium. 3 h later remove the medium, wash the cells with prewarmed medium and add 2 ml 20% glucose, 10% DMSO in HBS. Watch the cells under the microscope and, after no more than 4-5 min and before the cells shrink remove the DMSO solution, wash the cells twice in growth medium and return to the incubator with 5 ml fresh, prewarmed growth medium. Maximum expression of transfected genes is obtained 48 h after transfection when the cells can be harvested. Otherwise a selection system may be applied to obtain transformed clones (e.g. HAT to select for T K + cells - see 8 13.2). Bufyrute treatment (Gorman, 1985) The proportions of cells showing transient expression can be increased three fold (up to about 40%) by treatment with butyrate. Butyrate inhibits the deacetylation of histones and its effect may be to increase the proportion of transfected DNA assembled into active chromatin. About 4 h after transfection (and after the DMSO shock if included) add sodium butyrate to the medium and incubate overnight. The next day the butyrate containing medium should be removed and replaced with fresh, prewarmed growth medium. The concentration of butyrate should be between 2 and 10 mM and some preliminary experiments are required to determine the

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most appropriate level. Sodium butyrate solutions are prepared by neutralising butyric acid with NaOH, and they can be sterilised by filtration. 7.5.2. Liposornes

Lipofectin is the name of the liposome reagent available from Gibco BRL. It is a mixture of 5 mg DOTMA (N-[1-(2,3,-dioleyloxy) propyll-N,N,N-trimethylammoniumchloride) and 5 mg dioleoyl phosphatidylethanolamine sonicated in 1 ml distilled water. 1) Dissolve the DNA (1-1Opg - the amount must be determined for each cell type) in 1.5 ml HBS buffer (NaCl, 150 mM, Hepes, 20 mM pH 7.4). 2) Take 50-loop1 lipofectin and add it to 1.5 ml HBS buffer and mix it with the DNA solution. Complexes form immediately. 3) Use just-confluent cells and wash them twice with HBS buffer. Add the lipid/DNA mixture (3 ml per 100 mm plate). 4) Incubate for 5 h at 37°C and then add 10 ml growth medium. After a further 16 h replace the medium with 10 ml fresh medium. The ratio of DOTMA to DNA is critical but efficiency in immediate expression assays and in transformation is reported to be from 6-80-fold greater than with calcium phosphate or DEAE dextran (Felgner et al., 1987). Furthermore, the reagent can be used to transfect some cells which are refractory to other methods. 7.5.3. Electroporation

Electroporation is a technique in which the membrane of cells is reversibly permealised by a brief high voltage electrical discharge. It is often used for introducing DNA into cells when other methods are not successful. Thus, it has been used for introducing DNA into primary hepatocytes (Tur-Kaspa et al., 1986) and into lymphoid cells (Toneguzzo et al., 1986; Cann et al., 1988) and is the preferred method for plant protoplasts (Saul, M.W. et al., 1988). An overview

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of electroporation has been written by Shigekawa and Dower (1988) and the effective parameters reviewed by Knutson and Yee (1987). Uptake is proportional to D N A concentration in the range 30 ng to 10 pg per ml and the D N A can be supercoiled, linear or even single stranded (Bertling et al., 1987). Up to l o 4 D N A molecules are taken up by each cell and maximum intranuclear concentrations are achieved by 15 h. A number of commercial machines are available and cuvettes may be bought or constructed from disposable spectrophotometer cuvettes into which aluminium electrodes are glued. They can be sterilised with 70% ethanol and then washed with sterile PBS. (This is also true for the ‘disposable’ commercial cuvettes.) The same apparatus can also be used for cell fusion (electrofusion) (see 6 13.7.3 and Glassy, 1988). It is important to optimise conditions in order to achieve efficient electroporation. The important parameters are the voltage gradient, which should be up to about 4 kV/cm; the capacitance; and the resistance of the medium; all of which affect the shape and decay characteristics of the waveform (an exponential decay with a drop to 1/e (37%) in about 5 msec may be most effective). In general, a low field strength and a long decay constant seem to be most effective. High capacitance is advantageous, especially for lymphoid cells and, using a capacitance extender available from BioRad, this can reach 960pFd. Normal power packs can be used (Bertling et al., 1987; Knutson and Yee, 1987) but it is difficult to measure the actual voltage or waveform. The medium and the temperature are also important. If cells are left in buffer in the absence of nutrients at room temperature then viability drops rapidly. On the other hand, electroporation in serum-containing medium will introduce much more than the D N A into the cells although viability might be improved. Keeping the cuvettes on ice before and during the electroporation may also improve viability. Serum-free medium can be used at room temperature when viability is maintained and undetermined materials are not introduced into the cells. The optimal conditions for each cell need to be determined but variations on the following protocol are being used for transient

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assays for various lymphoid cell strains by Dr. Peggy Anderson in our department. 1) Harvest and sediment the cells (5 X lo6) by spinning at 1500 r.p.m. for 5 min in a universal at room temperature. 2) Decant the supernatant and resuspend the cells in 3 ml fresh, serum- free medium (RPMI). 3) Respin and resuspend the cells to 0.8 ml serum-free medium and add 1-4pg test DNA (from a 1 mg/ml solution in Tris HCl pH 8.0). 4) Transfer immediately to the cuvette and apply the optimal voltage pulse, e.g. 0.4 kV for an 0.4 cm cell at 960pFd (follow the detailed instructions supplied by the electroporator manufacturer). The voltage should drop to l/e in 11-16 msec *. 5) Transfer the cells immediately to a 25 cm2 flask containing 5 ml growth medium and return to the CO, incubator. 6) Harvest the cells 2 days later.

7.6. Cell visualisation A major advantage in the use of cell cultures is that they may be inspected constantly throughout the experiment. As healthy cells are almost transparent this is often done using phase microscopy. However, as the cells are growing on the bottom of a dish or bottle and the depth of the vessel is too great to allow a microscope to focus through it, microscopes had to be developed where the objective was below the microscope stage. Such inverted microscopes (Fig. 7.8) are available from most microscope manufacturers. Two important points to note when purchasing an inverted microscope are a) the lamp housing should be adequately ventilated for prolonged use in a 37°C hot room, and b) the distance between the microscope stage and the condenser should be sufficient to enable the largest roller bottle to be accommodated.

* Fibroblasts are effectively electroporated at higher voltage (4 V/cm) and lower capacitance (25gFd) when the voltage drops to l / e in 0.5 msec.

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Fig. 7.8. Inverted microscopes. From left to right, Wild, Olympus and Prior.

7.6.1. Phase contrast microscopy (Gray, 1972; Bradbuy, 1976)

Thts is extremely useful for examining living cell cultures which are almost transparent. Cellular material does change the vibrational state of the light and this may be detected using a phase microscope. The system is based on the phase alteration of light rays that are transmitted through the optical system of the microscope. 1) With the preparation on the microscope roughly in focus adjust the substage condenser to focus the iris diaphragm (partially closed), if necessary centering this to the field. 2) Focus the specimen. 3) Replace one eyepiece with the auxiliary telescope and focus this on the dark ring in the phase plate. 4) Superimpose the bright annulus exactly on the dark phase ring by centering the annulus holder. 5 ) Replace the normal eyepiece. Nomarski differential interference contrast microscopy is an alternative to phase contrast microscopy which gives an almost three

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dimensional image. For this technique it is essential to use planachromat objectives together with a Nomarski interference condenser and this is not available with an inverted microscope. 7.6.2. Fluorescence microscopy (Nairn, 1969)

T h s is commonly used when cell preparations have been treated with a fluorescent antibody (see 0 14.3.2) or with Hoesch 33258 when testing for mycoplasma (0 9.8.3). It is also used for chromosomes stained with quinacrine (see 8 7.4.3) and can also be used to visualise nucleic acids etc., by taking account of their fluorescence when excited by light of wavelength about 260 nm. It is important to use a powerful lamp which produces light of short enough wavelength. Commonly a mercury vapour lamp is used, though a certain amount of work may be done with the cheaper quartz iodide lamp. The light first passes through a primary filter which only allows through light of wavelength required to excite the fluorescence. When this light passes through the specimen some of it is excited to a longer wavelength. A secondary, barrier filter removes the exciting wavelength allowing only the light of altered wavelength to proceed to the eyepiece. In the more modern fluorescence microscopes the primary filter has been replaced by a system of mirrors (the Ploem illuminator in the Leitz microscope) which serves a similar function.

7.7. Subcellular fractionation The disruption of tissue culture cells and their subsequent subcellular fractionation may be more difficult than parallel processes with say rat liver. Although the cells are homogeneous they are usually small and cells grown in suspension frequently have very little cytoplasm. Disruption often requires the use of hypotonic media and, where possible, non-ionic detergents. For nuclear isolation a buffer containing 20 mM Tris HC1, 1 mM EDTA and 1%Tween 80 or Triton X-100 is suitable and dithothreitol and protease inhibitors may be

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included. This procedure leads to disruption of the cytosolic membranes. Cells, scraped from the growing surface or sedimented from a suspension culture are washed in PBS to remove serum and other unwanted medium components and then homogenised for example, using 5 strokes of a Potter-Elvehjem homogeniser or, for small volumes, by pipetting. Cell densities can be as low as lo6 cells per ml. Disruption is checked using a phase contrast microscope. The nuclei are readily sedimented at 800 g for 10 min. When isolating cytoplasmic organelles the use of detergents is not possible and hypotonic buffers should be avoided. If this is not possible then, immediately after homogenisation, tonicity should be restored by addition of a concentrated sucrose solution. The cytoplasmic organelles may be separated from each other by differential centrifugation or by density gradient centrifugation using sucrose or one of the commercially available alternatives, e.g. Percoll or Metrizamide. Some alternative approaches are considered by Howell et al. (1989). Attardi and Ching (1979) describe a method which has been used to reproducibly isolate mitochondria from cultured cells. Their method originally used Hela S3 cells growing in suspension: - Sediment the cells and wash with NKM (0.13 M NaC1, 5 mM KCl, 1 mM MgCl,). - Resuspend in 6 vol Tris acetate (10 mM, pH 6.7), KCl (10 mM), MgCl, (0.15 mM) and leave to swell for 2 min. - Homogenise using a teflon pestle rotating at about 1500 r.p.m. until 60-70% of cells are broken. - Add sucrose to 0.25 M and spin at 1200 g for 3 min to sediment unbroken cells, nuclei and large debris. - Sediment mitochondira by spinning the supernantant at 5000 g for 10 min. - Resuspend in 0.25 M sucrose, 10 mM Tris acetate pH 6.7, 0.15 mM MgCl, and respin at 1100 g for 2 min. Discard the pellet of debris. - Spin the supernantant at 5000 g for 10 min and resuspend the final mitochondria1 pellet in 0.25 M sucrose, 10 mM Tris acetate pH 7.0 at 2-4 mg protein per ml.

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CHAPTER 8

Glassware preparation and sterilisation techniques 8.1. General Cells in culture are fastidious. Not only do they require the absence of any toxic material in their growth media (which must be made up using high quality glass distilled water) but any glassware with which they or their growth media have contact must also be scrupulously prepared. It is largely for this reason that commercially available tissue culture plasticware is so popular as its use, in general, eliminates two, otherwise frequent, suspicions: 1) were the bottles and pipettes clean? and 2) were they sterile? Furthermore, as help from laboratory workers and technicians becomes ever less available and the price of plastics relatively lower, there are few who still use large amounts of glassware. Occasionally, however, most laboratories will find the need to recycle glass vessels. After use all contaminated glassware should be soaked overnight in baths of disinfectant. Highly recommended is chloros, a hypochlorite solution manufactured by ICI Ltd. and available in 5 gallon drums from McQuilken’s (Appendix 3) or Scottish Agricultural Industries, Renfrew. As this is an aqueous solution of sodium hypochlorite it is not suitable for metal caps and instruments which should be decontaminated by autoclaving. It is very caustic and care must be taken not to splash it onto clothes or exposed areas of skin. Chloros (1%) has the effect of killing any viruses, bacteria or cells which may be present, and at 0.25% also prevents proteinaceous materials from drying out and becoming irreversibly fixed to the glass surface. Uncontaminated glassware should be immersed in baths of water. It is usually convenient to have baths of 0.25% chloros or water 151

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placed strategically in the laboratory and these should be cleared once a day. The chloros must be renewed weekly as its effectiveness quickly wears off. After the indicated washing procedure all apparatus must be rinsed in deionised water before drying. Deionisation may be achieved by passing the water through a cartridge available from Elgastat Ltd., or Millipore Corp. (Appendix 3). It is important to maintain the cartridge in good condition and its effectiveness should be checked daily. 8.1.1. Washing procedure A number of different procedures are in use, but it is essential to avoid the use of abrasive and highly caustic cleaning agents such as Vim or concentrated acids. The alkaline sodium metasilicate is a very suitable cleaning agent and has the advantage that if a layer of metasilicate remains it is deposited as glass on neutralisation. Calgon metasilicate (CMS) is made up as follows. Dissolve 360 g sodium metasilicate (British Drug Houses, technical grade) and 40 g calgon (a water softener, available from R. & J. Wood, Ltd.) in 1 gallon of hot water. Dilute 1 to 100 with water for use. As an alternative to CMS the detergents Decon or 7X may be used. These come as concentrated liquids and are more convenient to use than CMS. Decon 90 (phosphate free) must be used in hard water areas but Decon 75 is suitable in soft water areas. Decon (available from Decon Labs Ltd.; Appendix 3) should be used as a 2% solution. 7X (available from Flow Laboratories; Appendix 3) should be used as a 5% solution. They are anionic surfactants. 8.1.2. Bottles and pipettes 1) Glassware is removed from the soaking baths and cotton wool plugs removed from pipettes (take care not to splash the caustic chloros). 2) Place calgon metasilicate (CMS) in a boiler and boil for 20 min. Alternatively bottles may be filled with hot CMS, Decon or 7X and left overnight.

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3) Allow to cool and rinse six times in tap water. 4) Rinse three times in deionised water. 5) Dry in a hot air oven - if any white streaks are found on the sides of the vessels the rinsing procedure is inadequate. This may be a result of failure to rinse sufficiently or it may indicate that the deionised water supply is faulty. 8.I . 3. Rubber stoppers

Natural or silicone rubber stoppers and capliners should be boiled in CMS, Decon or 7X, rinsed well in tap water and boiled in deionised water for 10 min. After further rinsing in deionised water they should be dried at low temperature. The metal caps should not be treated with CMS but may instead be boiled in a 1% bicarbonate solution. 8.1.4. Glass washing machines

If a suitable glass washing machne is available then after stage (2) described above, the glassware may be loaded into the machine for a further wash and rinsed using deionised water. Addition of detergent at this stage is usually unnecessary but if required powdered Decon or 7X-omatic may be used. 7X-omatic (Flow Laboratories; Appendix 3) is a low foaming liquid containing a non-ionic surfactant and should be used as a 1% solution.

8.2. Sterilisation by heat The following are methods of sterilisation: Hot air Autoclaving Filtration Irradiation, e.g. 60Co y-irradiation is used commercially for disposable plastics Ethylene oxide treatment

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The last two methods are not generally applicable to the small laboratory except insofar as low pressure mercury vapour lamps emitting light of 254 nm may be used to sterilise the air in aseptic rooms and cabinets (see 9 9.4.1). 8.2.1. Hot air

This involves heating the glassware to 160°C for 90-120 min. It is the preferred method for bottles which do not have screw caps when their orifice should be covered with aluminium foil. It is also used for glass Petri dishes and pipettes. Petri dishes should either be stacked in tins or containers specially designed for them or simply held closed with sterilisation tape. This brown paper adhesive tape has pale strips which turn dark brown under sterilising conditions and is used as an indicator of the effectiveness of the procedure. It is available from the 3 M Company Ltd. (Appendix 3). Pipettes are plugged with non-absorbent cotton wool and placed in aluminium or steel cans (obtainable from Gallenkamp or A. & J. Beveridge; Appendix 3) for sterilising. At the bottom of the can a pad of cotton wool may be placed to prevent damage to the pipette tips. This pad should be renewed each time the pipette can is sterilised as prolonged heat causes the cotton wool to carbonise producing material toxic to cells. A Browne’s steriliser tube Type I11 (see below) should be placed inside the central can of each batch being sterilised. 8.2.2. Autoclaving

Heat-stable solutions, rubber bungs and liners, bottles with plastic caps, ultrafiltration apparatus etc. are all .sterilised by steam treatment at elevated pressure. Although the time required to sterilise is usually only about 15 min at 15 lb pressure the cycle time for modern autoclaves is several hours. This is because of the safety precautions built into these machines to prevent the doors being opened until the temperature of liquid within bottles has fallen to 80°C.

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To sterilise small items such as rubber bungs they should be placed in glass Petri dishes and wrapped in aluminium foil. Empty bottles should have their plastic caps only loosely screwed on to allow penetration and escape of steam during the sterilisation cycle. Partly filled bottles should have their caps firmly screwed on. The steam generated within the bottle will effectively sterilise the contents but changes in volume will be prevented. It is such bottles whch create the hazard in autoclaves if the temperature and pressure outside the bottle is allowed to fall suddenly. This will happen if the autoclave door is opened prematurely leaving the bottle itself full of superheated steam. To obtain sterile gauze for filtering trypsinised suspensions when setting up primary cells the gauze is best arranged in a filter funnel and the whole wrapped in aluminium foil so that it can be unwrapped without contamination. Such wrapped funnels are sterilised by autoclaving. Filtration apparatus (see below) can be assembled with the filters in position and the whole autoclaved. It is important either to plug orifices with cotton wool or to cover them with aluminium foil caps. When it is desired to sterilise only a small amount of material the use of an autoclave may be extravagant. In this case an ordinary pressure cooker with facilities to go up to 15 lb pressure may be used, but precautions must be taken to allow adequate time for cooling before the cooker is opened. 8.2.3. Control of sterilisation Browne’s steriliser tubes contain a liquid that undergoes a slow chemical reaction at elevated temperatures, changing from red to amber then green. Only when they are green has the correct temperature time combination elapsed. Several of these indicator tubes should be distributed in each batch of material to be sterilised. It is important when autoclaving liquids, to place a Browne’s tube in a bottle of liquid as the conditions within such a bottle differ from those outside. Different types of Browne’s tubes are available for sterilisation using steam or dry heat. Type I (black spot) tubes are

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for use for steam sterilisation and type I11 (green spot) for sterilisation by dry heat. They are available from Albert Browne Ltd. (Appendix 3) and each box comes with instuctions for use and a colour code.

8.3. Sterilisation by filtration Membrane filters remove, from a liquid passing through them, all particulate matter larger than the filter pores. They are of high porosity giving reasonably high flow rates (up to 12 ml water/min/cm2 at 10 p.s.i. for a 0.2 pm filter) and by selection of the appropriate pore size the resulting filtrate is rendered sterile. It is usual to have a 0.22 pm pore, though in some cases smaller pores are required and often two or even three membranes of decreasing pore size are used, e.g. 0.45, 0.22 and 0.10 pm. The filters are made of cellulose esters or aromatic polymers and may be sterilised by autoclaving. They are first fitted into a filter holder and where necessary covered in aluminium foil. They are available in a large range of diameters and so filtration is a convenient method of sterilising volumes of liquid ranging from less than 1 ml to 5 gallons or more. 8.3.1. Self-assembledfilter apparatus

For small volumes, 13 or 25 mm diameter, filtration membranes may be fitted into plastic or stainless steel holders (e.g. the Swinnex filter holder made by the Millipore Corp.; Appendix 3) which, after autoclaving, are fitted onto a syringe containing the liquid to be sterilised. Care must be taken in the assembling of the membrane in the holder as incorrect assembly leads to the escape of the membrane from its retaining gaskets with subsequent failure of the filtration process. The plastic holders have a limited life time as they distort on autoclaving. For volumes up to about 300 ml stainless steel filter holders are available (see below). These take a 47 or 50 mm diameter membrane filter and incorporate an upper chamber which holds either 100 ml

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second glass b e l l

g+

100 rnl reagent bottle

Fig. 8.1. Diagrammatic outline of the arrangement of vessels used in filter sterilisation. When filters of larger diameter are used the filter holder is not designed to hold any liquid and a dispensing pressure vessel must be used. However, with the 100 ml filter holders the dispensing pressure vessel is unnecessary if only small volumes are to be filtered. In this case the filtrate may be collected directly into a sterile reagent bottle.

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or 330 ml of the liquid to be sterilised. Once assembled the unit is sterilised by autoclaving with the lower outlet covered with aluminium foil. In use the unit is clamped in a vertical position with the outlet over a sterile receiving vessel. The liquid to be sterilised is placed in the upper compartment and pressure is applied to force the liquid through the membrane. The pressure may be applied from a cylinder of compressed air or by means of a small air pump. Using a 0.22 pm membrane filter and a pressure of 10 p.s.i. a flow rate of 140 ml/min can be achieved. To reduce the chance of contamination a neoprene stopper may be fitted to the outlet tube and this fitted into a Buchner flask with a cotton wool filter fitted to the side arm. The whole assembly may be autoclaved together. Another method is to fit a 'bell' to the outlet tube (Fig. 8.1). The volume of liquid that may be filtered through such a filter can be increased by coupling the holder to a dispensing pressure vessel. Vessels holding up to 20 1 may be interposed between the smaller filter holder and the supply of compressed air so that liquid is constantly transferred from the large reservoir to the small reservoir above the membrane. However, a 47 mm hameter filter often clogs up before filtration of such large volumes is complete and it is recommended that a large, e.g. 142 mm diameter, filter is used (Fig. 8.2.). 142 mm diameter filter holders do not have a built-in reservoir and must be coupled to a dispensing pressure vessel or medium must be pumped under pressure into the filter. However, flow rates of up to 1.5 l/min can be obtained with a pressure of 10 p.s.i. and the holder comes fitted with legs which conveniently can sit astride the collecting vessel. These stainless steel filter holders are expensive and require practice in order to assemble them correctly. They also require careful washing and sterilising. If it is necessary to refill the dispensing pressure vessel during filtration it is important that the pressure above the membrane filter does not fall or contamination may result. To avoid such a pressure fall valves may be fitted. Foot operated systems are available from Schleicher and Schuell which allow the flow of liquid to be interrupted at regular intervals; this is very useful when filling 100 ml bottles from an otherwise continuous flow of 10 1 of sterile medium. However, we have found the system cumbersome to use and we

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Fig. 8.2. 142 mm filter holder assembled to filter medium contained in the steel pressure vessel. Pressure is generated by the pump on the right. The sterile medium is collected into bottles held under the bell below the filter which is clamped between the two steel plates.

prefer to collect the sterile medium first into a large, sterile bottle and subsequently withdraw 100 ml aliquots using a rubber hose and a second filling bell (see Fig. 8.1). Although the Millipore Corporation offer a very wide range of filter holders and accessories, other companies provide valuable competition: e.g. Anderman & Co., Ltd. (Appendix 3) are agents for Schleicher & Schuell who manufacture a comparable range of filters and holders etc., and V.A. Howe supply Sartorius membranes and filter holders. 8.3.2. Disposable filter apparatus Although superficially more expensive, the use of a disposable filter apparatus is far more reliable than the self assembled varieties, which are not recommended for the occasional user.

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Fig. 8.3. Disposable filters. The small Millex filters (a) and the larger Gelman Acrocap (b) can be attached to a syringe or used on line. In both cases positive pressure is used to force the medium through the filter. With the Nalge and Falcon filters (c) negative pressure from a water pump sucks the filtered medium either into the base of the filter or into a receiving vessel. (Courtesy of the manufacturers.)

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Fig. 8.3 (continued).

For filtration of small volumes (5 -20 ml) disposable filter units are available which can be attached to a disposable syringe (Fig. 8.3). The contents can be expressed directly into a sterile receiving bottle. Such filter units are available from Millipore (Millex Filters), Schleicher and Schuell or Gelman (e.g. Acrodisc 13: Appendix 3) (Fig. 8.3). A 37 mm diameter filter in a similar holder can be used on-line to filter up to a litre of serum-free cell culture medium. Millipore’s Sterivex is a tangential flow filter which can be used with a repeating syringe or on line to filter up to 1 litre of growth medium and Gelman’s Acrocap (Fig. 8.3) is similar. Sterile disposable units (capacity 120-500 ml) are available from Nalge, Falcon and Gelman. These units resemble a sterile beaker to

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Fig. 8.4. Larger volumes can be filtered through a Gelman Micro Culture Capsure (a) or a Millipak filter (b). These can be organised in a manner similar to the 142 mm filter (Fig. 8.2) or medium may be pumped directly onto the filter as shown in (b). (Courtesy of the manufacturers.)

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163

Fig. 8.4 (continued).

the top of which is sealed the filter. A reservoir fits above the filter. A vacuum line is attached to an outlet on the side of the receiving beaker so that filtration is under negative pressure (see Fig. 8.3). Because of this it is not very suitable for filtration of proteinaceous solutions. It is a very convenient method, however, of filtering protein-free solutions and the filtrate is obtained in a portable form which is not readily contaminated. Alternatively filtration can be into a bottle on top of which the filter sits. Other disposable filter units are available for filtration of large volumes of cell culture medium. Millipak filter units (Millipore Corp.; Appendix 3) are available in various sizes to filter up to 150 1. The units are presterilised and come with a filling bell. Medium can be pumped directly into them (Fig. 8.4). Gelman’s ‘culture capsules’ give effective filtration areas of 300 and 500 cm2 (and larger ones are available) by providing a corrugated filter sealed into a plastic housing so that the filtration takes place at right angles to the overall

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direction of medium flow (tangential flow filter). The smaller unit which comes with attached filling bell (Fig. 8.4) is suitable for filtration of 10 1 of cell culture medium and simply needs to be connected to a dispensing pressure vessel or pump (see 8 8.3.1). It has a side vent in case air bubbles accumulate. It costs about E l l (1989 prices) which adds over f l per litre to the cost of ‘home produced’ medium but in terms of its labour saving potential is good value. This is one of the hidden costs when evaluating whether to prepare one’s own medium or to buy it already made at X 1 or X 10 concentration - see 8 5.3. Some of these disposable filter units are shown in Figs. 8.3 and 8.4 and filter units are available for even larger volumes, e.g. MilliPak-100 will cope with up to 75 1 serum-free medium at a flow rate of 35 1 per min. It has been reported recently (Knight, 1990) that solutions filtered through disposable filters may be detrimental to the growth of some cells. This is because the filters are treated, during manufacture, with polyglycolethers. These may be removed by washing the filter before use with warm, sterile distilled water followed by a small volume of culture medium.

CHAPTER 9

Contamination 9.I . Bacterial contamination All culture media provide an excellent environment for the growth of microbial contaminants, and average cell culture procedures (involving the frequent opening of culture vessels) provide ample opportunities for contamination. Moreover, whereas the human body has multiple defences against infection, a bottle of cells relies on the skill of the manipulator. Although antibiotics are commonly added to short-term cultures they are not recommended for long-term cultures. It is essential that stringent precautions are taken to exclude microorganisms and that adequate tests are performed to ensure that the precautions have been effective (see 0 9.2). 9.1.1. Glass- and plasticware

The methods of preparation of glassware are indicated in Chapter 8, and if sterilisation is monitored as described the glassware should not be a source of contamination. Likewise plasticware is obtained from the manufacturer in a sterile condition. Usually sterilisation of plastic is achieved using ethylene oxide or irradiation procedures and vessels are supplied wrapped in cellophane. 9. I . 2. Cells

Contaminating microorganisms may be present in the cells when these are obtained. The procedures for obtaining primary cells should ensure the exclusion of contamination, but all cells entering the laboratory should be tested before carrying out transfer experiments in rooms being used for other cell transfers. 165

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9.1.3. Media

Certain items, such as balanced salt solutions and versene which are heat-stable, are generally sterilised by autoclaving, but the majority of organic materials used in cell culture are filter-sterilised. For heat-sterilised materials it is generally sufficient to rely on a sterilisation indicator which should be included with each batch of materials being sterilised. Often autoclave tape is sufficient if used on each packet, but for solutions and larger containers, e.g. cans of pipettes, it is recommended that a liquid indicator is included within the bottle or can.

9.2. Sterility checks Sterility checks should be made on all batches of media and trypsin before these are used. As some tests take several weeks to complete, it is essential that adequate stocks are maintained. The use of untested medium is a sure way to introduce contamination into all cell lines in use in a laboratory. Similarly, cell cultures should be routinely checked for contamination. Such checks will involve growth tests on the medium in which the cells have been growing as well as tests on the cells themselves. No single test is sufficient to detect all possible contaminants and hence multiple procedures must be adopted. Organisms that grow rapidly in cell culture medium are readily apparent when contaminated medium is incubated at 37°C for a few days. Such contamination is not a serious problem as the experiment can quickly be terminated and the contaminated culture eliminated. It is the slow-growing contaminant which produces no obvious change in the medium and which exerts no marked cytotoxic effect, which may be overlooked and yet may dramatically interfere with a biochemical investigation, e.g. satellite DNA bands in CsCl density gradient analyses or unusual forms of enzyme may reflect the presence of a contaminant . In order to detect slow-growing or fastidious contaminants a sample (usually 0.5-1.0 ml) of the culture medium, cell suspension

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TABLE9.1

Thioglycollate broth Sabouraud’s medium Blood agar plate Deoxycholate plate

Temperature 20°C 37°C

Aerobic

Anaerobic

+ +

+ + + +

+ +

+ + + +

or trypsin solution is inoculated into a series of broths (20 ml) or smeared on nutrient plates. The size of the inoculum may be limiting in the case of very low levels of contamination but is chosen for convenience. Armstrong (1973) recommends that all freshly prepared media etc., should be tested for possible contamination prior to addition of antibiotics. - Incubate samples at 37°C for 1 week. - Remove duplicate 1 ml aliquots into a) thioglycollate broth, and -

-

b) Saboraud’s liquid medium (Appendix 4). Also streak samples onto two blood agar plates (Appendix 4)and two deoxycholate plates. Incubate as shown in Table 9.1.

Various other broths for detecting bacterial and fungal contamination include brain heart infusion broth, tryptose phosphate broth and trypticase soy broth. These should be made up as per manufacturers’ (Oxoid Ltd. or Difco Labs.; see Appendix 3) instructions, but some procedures are given in Appendix 4. Incubation times for the various tests should be at least 7 days, although all but the least obvious contaminations will show after 2 days. The temperature of the incubations should be at both 37OC and at 30°C or 20°C. It is important to maintain the agar plates in a sealed container or humidified incubator to prevent their drying out. Containers are available commercially which not only maintain humidity but also allow anaerobic conditions to be maintained by ensuring the removal of all traces of oxygen. Should a precipitate

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form in a broth culture, it should be examined microscopically to determine its nature. Such manipulations, however, must be carried out at the end of the working day and must be followed by severe decontamination of the work area and surrounding air space.

9.3. Analysis of bacterial contamination Using an aseptic technique remove a sample of ‘spent’ medium and sediment it at 20,000 g for 20 min. A loop of the sediment should be spread on a blood agar plate (Appendix 4) and incubated at 37°C for 2-7 days when most bacterial contamination will show up. Samples should be smeared onto glass slides and air-dried prior to staining with ‘Gram stain’ or methylene blue (Appendix 2).

9.4. Airborne contamination Despite what has been said above, the major cause of contamination is inadequate aseptic technique allowing organisms from personnel to enter cultures. Such airborne contamination may occur any time a culture vessel is opened. Consider that if 10 ml of a cell suspension is removed from a vessel it is replaced by 10 ml of air. It is therefore essential to reduce airborne contamination to a minimum. In an undisturbed room bacteria and fungal spores rapidly settle to the floor or the bench, and hence regular cleaning of the floor and bench with antiseptic solutions is required. The floor of the work room should be free of cracks and should be cleaned daily with a disinfectant solution. The work bench should be swabbed down before and after each use with a solution of 70% ethanol. This also serves to kill cultured cells which may have been spilt and hence prevents their transfer to other cultures (see 0 2.2). It is also recommended that the air supply to transfer rooms is filtered through high efficiency particulate air (HEPA) filters which

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169

remove virtually all particles larger than 0.3 p m diameter and hence sterilise the air entering the work room. When an aseptic room is not in use the air may be sterilised by use of UV germicidal lamps. These should be installed in a position such that the whole room is illuminated and should be sufficiently powerful to be effective at the extreme corners of the room. Many of these preventive measures are made much easier if, instead of a small room, a cabinet is used to carry out aseptic transfer (see below). In addition to these precautions the necks of all bottles and pipettes etc., should be ‘flamed’. This does not have the function of sterilising the glassware but of raising its temperature above the ambient and thus causing an upflow of air around the bottle or pipette. It prevents airborne contaminants from settling into flasks or onto pipettes. 9.4.1. Aseptic technique

The details of a good aseptic technique cannot be written in a book. ThFy must be demonstrated and practised assiduously under the guidance of a skilled operator. It is, however, largely a matter of common sense, and a few do’s and dont’s are listed below. -

-

Always use plugged pipettes and preferably keep your head as far from the open vessels as possible. This can be done by using a mouthpiece and a short rubber tube to the pipette. However, a much more convenient, if more expensive method involves the use of a pipette-aid (Fig. 9.1). These are sold by a number of companies (e.g. Camlab, Jencons, Laser, Northumbria, Flow Labs; Appendix 3). The cheaper models have a hand-operated pump and are tiring to use. Mains operated models require a wire stretching across the work bench which is awkward and can knock over bottles. The best type are rechargeable and the more modern versions are very light and easy to use. Use automatic pipettes (e.g. Finnpipette, Gilson, Oxford) and sterile tips for small volumes. Repeating versions and eight place

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Fig. 9.1. Pipette aids. The figure illustrates three types of pipette aid which can reduce the chance of contamination. In the centre is a manual model which requires pumping. Although cheap, this model can be tiring to use. The mains operated model at the bottom has the problem of a wire which can interfere with manipulations. The rechargeable battery operated model at the top is preferable and it works for 5-20 h on a single charging. (Courtesy of Flow Laboratories.)

CH. 9.

-

-

-

-

-

-

CONTAMINATION

171

pipettes are available for filling microtitre plates though it is often difficult to get all eight tips firmly attached to these devices. Always work in a laminar flow cabinet (5 9.4.2) or close to a bunsen flame. Keep the tip of the pipette pointing forward (do not tuck it under your arm) and do not handle it on the graduated part. Loosen bottle caps before choosing a pipette. It is then a simple matter to raise the lid with one hand, pipette with the other, and replace the lid. This avoids the difficulty of removing a lid with the little finger of the pipetting hand. When withdrawing a pipette from a can, do so without touching any other pipettes. This can be done by shaking the can until a few pipettes protrude from the opening. If a second pipette is accidentally touched, it should be removed immediately. Replace the lid on pipette cans and bottles as soon as practicable. Discard all used glassware which may be a source of contamination into baths of disinfectant (chloros). Never leave dregs of medium around to provide a source of bacterial nutrient. If you drop a bottle cap - get a new sterile one. All bottles of medium prewarmed by standing in a water bath 'should be thoroughly dried with tissues (not a dirty towel) before being taken into the aseptic transfer room. Water baths should contain bacteriostatic agents, e.g. Panacide (British Drug Houses Ltd.; Appendix 3). If in doubt decontaminate the outsides of all bottles with a tissue soaked in 70% ethanol.

If all the above precautions are taken, then the only remaining source of contamination is the person working with the cells. Personnel carry microorganisms on the clothes and on their person, especially on their hair. It is a good idea to wear a clean laboratory coat restricted to use in the cell culture laboratory and never to move from an animal house into a cell culture laboratory without changing laboratory coats. People with long hair should tie it back and wear a clean head covering. It is also recommended that face masks are used especially if there is any doubt about a person's health.

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9.4.2. Laminar flow systems

Laminar flow cabinets serve two purposes; to protect the samples and to protect the worker and the environment. For most tissue culture applications sample protection is sufficient, but increasingly often we are becoming aware of hazards associated with biological samples and the cabinet to choose combines both aspects of protection. Such are the vertical laminar flow cabinets available, for example, from Flow Laboratories (Gelaire) or M.D.H. (Appendix 3). In the cabinet with vertical flow, a stream of air, filtered through a HEPA filter, enters the cabinet through the ceiling and passes in a piston-like manner past the working surface and out through the perforated floor of the cabinet. Air entering the front of the cabinet prevents aerosols leaving by that route. Thus laboratory air is drawn out through an exhaust grill in front of the cabinet before reachng the working surface. The exhaust air is recirculated through the filter and back into the cabinet or out through a filter at the top (Fig. 9.2). When working with potentially pathogenic cultures it is essential that hazardous aerosols are removed by a viricidal filter before the air is vented to the general atmosphere. Horizontal-flow cabinets are satisfactory for aseptic manipulations such as media preparation where potentially hazardous aerosols are not generated. In these the filtered air enters through the back of the cabinet and leaves through the front. A horizontal-flow cabinet is a miniaseptic room which is much easier and cheaper to maintain than a walk-in aseptic room. In addition, it has the advantage that the worker is not confined within a small room which may become extremely hot and stuffy and, secondly, that the worker’s head is separated from the cultures by a perspex screen thus further reducing the chance of contamination. The use of a laminar flow system would appear to make it impossible for bacteria to pass from the worker to the culture and in principle they provide ideal protection for both cultures and workers. However, the very act of putting the hands into the air stream disturbs the laminar flow and causes eddies. For this reason laminar flow cabinets should only be used in addition to standard aseptic technique although it is unnecessary to work near a bunsen flame.

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173

Fig. 9.2. A photograph of a class 2 flow cabinet is shown (courtesy of MDH). The air flow diagram is drawn onto the photograph. Air entering at the front is immediately drawn under the work area and, together with air from the cabinet it passes up a space in the back of the cabinet to reenter the top of the cabinet through a HEPA filter.

9.5. Antibiotics The use of antibiotics in stock cultures is strongly discouraged. The relaxation in aseptic technique resulting from a reliance on antibio-

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tics leads to considerable contamination. The growth of the contaminants may be kept in check by the antibiotics, but biochemical alterations may still be produced. Under the selective conditions antibiotic resistant microorganisms will be selected and once established these may spread like wildfire through all the cultures in a laboratory. The use of antibiotics should be restricted to short-term experiments. These often involve a large increase in the cell number over a period of a week or so and a transfer of large numbers of cells, and their subsequent experimental manipulation is particularly difficult to perform aseptically. At the end of the experiment such cultures should not be returned to stock but should be treated as contaminated. Table 5.1 lists the antibiotics sometimes used to treat cell cultures.

9.6. Disposal of contaminated material No obviously contaminated vessel should be opened. Rather it should be autoclaved immediately. If it is desired to test the nature of the contamination, a sample should be taken aseptically to avoid the spread of infection. This is best done at the end of the working day, and the work area then thoroughly disinfected and the UV lamp left on overnight. Contaminated dishes should be placed in special disposal bags (Sterilin Ltd.) before autoclaving, and often it is advisable to decontaminate the incubator by swabbing with disinfectant. Contaminated cultures which are also radioactive may be autoclaved (dishes should be placed in disposal bags) and then treated as radioactive waste.

9.7. Mycoplasmas (Smith, 1971) These are small prokaryotic cells (0.3-0.5 pm in diameter) whxh can form small colonies resembling those of the agent causing bovine pleuropneumonia - hence their alternative name of pleuropneu-

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175

Fig. 9.3 Colonies of M.arginini growing on PPLO agar.

monia-like organisms or PPLO. The agents do not possess a cell wall and hence will only grow in certain media. When grown under agar, colonies resemble fried eggs in having a thickened central region (Fig. 9.3). There are several serological groups of mycoplasmas from two genera ( Mycoplasma and Acholeplasma). Those infecting cell culture come almost entirely from four species: M . orale (human), M . hyorhinis (pig), M . arginini (bovine) and A . laidlawii (bovine and also found in rodents). Although primary cell cultures are usually free from contamination by mycoplasmas, many cell strains and lines in use are contaminated. This probably arose from the use of animal sera contaminated with A . laidlawii and M . arginini or pig trypsin contaminated with M . hyorhinis. These are probably no longer a significant source of contamination and, although M . hominis, M . pharyngis and M . salioarium are readily isolated from human mouths and throats, the most frequent source of contamination today is from working with contaminated cells. It has been estimated that 50-95% of cells in use today are contaminated with mycoplasma.

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As mycoplasmal contamination of cell cultures is not always so obvious as bacterial contamination, it is important to 1) be aware of the effects of mycoplasmas on cell cultures, and 2) carry out routine tests for their presence. This is especially important as a contaminated culture may have 10' mycoplasma per ml, i.e. there may be 100 mycoplasma per cell. The mycoplasma often grow attached to the surface of the cell providing it with a prokaryotic coat.

9.7.1. Effect on cell cultures

The effects will depend to some extent on the species of mycoplasma involved. Thus while M . gallisepticum and M . mycoides are pathogenic they are very uncommon, and other species do not produce cell death, but retard the growth of cultured cells. In some cases this effect on growth rate can be attributed to arginine deficiency brought about by high levels of arginine deaminase in, for example, M . hominis (Kenny and Pollock, 1963; Kenny, 1973). A . laidlawii rapidly cleaves deoxribonucleosides (Hakala et al., 1963). Thus attempts to incorporate tritiated thymidine into such contaminated cultures are unsuccessful as the nucleoside is rapidly converted to thymine. 9.7.2. Culture of mycoplasmas

Mycoplasma show complex growth requirements (Rodwell, 1969) and hence do not readily grow in simple media such as Eagle's unless growing animal cells are also present. The effect has been attributed in part to the degradation of the DNA of the animal cells by deoxyribonucleases secreted by the mycoplasmas. The liberated nucleosides are essential growth factors for the mycoplasmas (Stock and Gentry, 1971; Stambridge et al., 1971; Kenny and Pollock, 1963). It is therefore important when attempting to grow mycoplasma to use media enriched in, for instance, yeast extract which supplies all essential nutrients. In addition, some mycoplasma grow better under anaerobic conditions.

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177

Kenny (1973) recommends a Soy peptone-yeast dialysate medium (Appendix 4) for culture of mycoplasma. Inclusion of arginine (16 mM) and 0.4 mg% phenol red indicates the presence of arginine deaminase by formation of alkali (purple coloration). Alternatively, incubation with tritiated thymidine and analysis of the culture medium for tritiated thymine can be used to detect thymidine phosphorylase (House and Waddell, 1967). A simpler medium based on the formula of Hayflick (1965b) is in use in our laboratory. 70 parts 3% brain-heart infusion (Difco Labs) 20 parts horse serum (preheated to 56°C for 30 min) 10 parts autoclaved yeast dialysate (or extract) supplemented with 0.5% glucose, arginine HCl and phenol red together with penicillin G (10,000 U/ml) and thallium acetate (0.05%). A more complicated growth medium is recommended by Barile and McGarrity (1983) 1 ml of animal cell suspension is inoculated into 10 ml broth medium. More often, for diagnostic purposes, however, PPLO checks are carried out using PPLO agar (Appendix 4). Culture of PPLO. It is important to use both a cell suspension and the culture supernatant from cells grown for 3 days. Take up the sample in a Pasteur pipette. Pierce duplicate PPLO agar plates with the pipette about 10 times each or simply cover the agar with the culture supernatant. Incubate in 5% CO, in either N, or air in a sealed jar, i.e. anaerobically or aerobically. Colonies appear in 3-7 days but incubation should be continued for 3-5 weeks when the colonies assume a typical ‘fried egg’ appearance (Fig. 9.3). Yeast extract and the various broth and agar media are available commercially from Oxoid Ltd. or Difco Labs (Appendix 3).

9.8. Testing for mycoplasma Observation of a monolayer of contaminated animal cells sometimes gives early warning of the presence of mycoplasma. Thus cells

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stained with haemotoxylin-eosin show the cytopathic effect characterised by multiple dark staining regions (granules). Although staining and autoradiography can be used, most commonly mycoplasma are detected using a fluorescent stain (9.8.3) in combination with growth on agar (9.7.2). A new test (9.8.4) is also very effective and picks up 97%of contaminants (McGarrity et al., 1986). 9.8.1. Orcein stain Fogh and Fogh (1968) give details of a method using orcein to detect mycoplasma. Seed cells onto coverslips so that they are not quite confluent after 48 h. - Put a coverslip of cells into a Petri dish containing 3 ml 0.6% sodium citrate and slowly add 1 ml of distilled water. Leave for 10 min and then slowly add 4 ml Carnoy’s fixative (Appendix 2). Pour off the fluid and replace with 2 ml Carnoy’s fixative. - Allow 10 min for fixation. The coverslip is then allowed to stand for 5 min or until absolutely dry. Stain for 10 min with orcein stain (Appendix 2) or longer if necessary and wash 3 times in absolute alcohol. - All the procedures are carried out at room temperature. - Examine using the phase microscope - infected cells show mycoplasma.located primarily at the cell borders and apparently in part attached to the peripheral parts of the cytoplasm and in the intracellular spaces. -

9.8.2. Autoradiography

Autoradiography of contaminated cultures labelled with tritiated thymidine shows grains over the whole cell rather than localised to the nucleus. This is a result of degradation of the thymidine to thymine followed by incorporation into mycoplasma DNA and other cell constituents. -

Seed cells at 2.5 X lo4 per 35 mm plate containing a c,overslipand label for 30-46 h with 5 pCi [3H]thymidine per plate.

CH. 9.

-

-

CONTAMINATION

179

Fix cells with ethanol : acetic acid (3 : 1) for 10 min in the dish. Repeat and wash twice with absolute ethanol. Mount the coverslip on a glass slide - cells uppermost - and dip into Ilford L4 emulsion (see 0 12.4.4). After 5-7 days develop in Kodak D19b developer and fix. Examine under the microscope. Cells free of PPLO show heavy nuclear labelling, whereas infected cells show cytoplasmic labelling with little or no nuclear labelling.

9.8.3. Fluoresence staining

The presence of mycoplasmal DNA in the cell cytoplasm or attached to the cell membrane may be detected by staining with the fluorochrome Hoechst 33258 (Appendix 3). This intercalating dye fluoresces under ultraviolet light and this forms the basis of a very rapid test for mycoplasmas. The fluorescence consists of a diffuse series of small granules apparently throughout the cytoplasm of infected cells. Confusion may arise from much larger, brightly staining fragments of nuclei which sometimes contaminate a culture (McGarrity et al., 1983). A stock of the Hoechst 33258 bisbenzamid fluorochrome solution is made by dissolving 5 mg in 100 ml PBS-A (Appendix 1) using a magnetic stirrer. It must be free of bacterial contamination and should be sterilised by filtration through a 0.22 pm membrane and should be stored in the dark at 4°C. It should be diluted 100-fold with filtered PBS-A for use. Set up coverslip cultures of the cells to be tested and use when 50-70% confluent. This is conveniently done using the Labtek 8 place culture chamber slide. - Aspirate the medium from the cells and fix with two changes of ethanol :acetic acid (3 : 1) over 10 min. - Wash in deionised water and incubate for 30-60 min at 37°C with diluted bisbenzamid fluorochrome. - Rinse with deionised water and mount the coverslips - cells down - on a slide using glycerol mountant (22.2 ml 2.1%citric acid; 1 H,O; 27.8 ml 2.8%disodium hydrogen phosphate; 50 ml glycerol pH 5.5) -

180

-

CELL CULTURE FOR BIOCHEMISTS

Examine with a fluorescence microscope when the presence of mycoplasma shows up as a cytoplasmic fluorescence (Fig. 9.4.).

Russel et al. (1975) report a similar staining method using 4’,6-diamidino-2-phenylindole (DAPI):

Fig. 9.4. Fluorescence staining for mycoplasma. Cells were stained with Hoechst 33258 which fluoresces with DNA. (a) Vero cells infected with M. arginini which show marked extranuclear fluorescence; (b) negative control cells. The photographs ( ~ 4 0 were ) kindly supplied by Dr. Rae of Moredun Animal Health Ltd.

CH. 9

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181

Fig. 9.4 (continued). -

Rinse cells with PBS 0.1 pg/ml at 37°C. Although staining is faint after 15 min, it is easier to distinguish cytoplasmic fluorescence with shorter incubation times than after 60 min when nuclear fluorescence is intense. Rinse with PBS and mount the coverslip as described above. This method, using unfixed preparations, is even more rapid than the one described above using Hoechst 33258. A kit based on DAPI is available from Bioassay Systems.

- Stain with DAPI at

-

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CELL CULTURE FOR BIOCHEMISTS

Autoradiography and staining methods are based on detection of cytoplasmic DNA (in the former case replicating cytoplasmic DNA). For this reason there will always be a low background caused by mitochondria1 DNA and samples to be tested should be compared with positive and negative controls. Such prefixed controls are available from Moredun Ltd. (Appendix 3) ready for staining. Difficulty is encountered in detecting mycoplasma in certain cell lines and, for this reason, indicator cells e.g. mouse 3T3 cells are often used. 24 hours after establishing a cover slip culture of 3T3 cells a sample of the supernatant of the cells under test is added to the culture. Positive and negative controls should also be included. Three to four days later cultures are tested using fluorescence staining and another technique. 9.8.4. Cell growth test for mycoplasmas

The lethal effect of mycoplasmas on cell cultures can be accentuated by culture in the presence of 6-methylpurine deoxyribonucleoside (6-MPDR) and this forms the basis of the ‘MycoTect’ kit available from Gibco. All mycoplasmas possess high levels of adenosine phosphorylase which converts the non-toxic 6-MPDR into 6-methylpurine and 6-methylpurine ribonucleoside, both of which are toxic to mammalian cells (McGarrity and Carson, 1982). Grow the cells in antibiotic free medium for two passages and harvest when nearly confluent by scraping the cells into the medium. - Allow the cells to settle out and use the supernatant (still containing a few cells) in the test. - Seed a 24-well plate with 2 X lo4 control, mycoplasma-free cells growing in 1.5 ml antibiotic-free medium per well. Gibco suggest the use of 3T6 cells. Allow to attach for 2 h. - Add 0.2 ml test sample to a well and incubate for 24 h. It is suggested that some wells are left as negative controls and to others 0.2 ml of a solution of adenosine phosphorylase is added to act as a positive control. This still leaves room to test five different cell lines using the single 24-well plate. -

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183

After 24 h add 50 p1 6-MPDR (final concentration 30 pM) to duplicate test and control wells and continue the incubation for a further 3-4 days when the negative control wells should be confluent. The positive controls and those wells containing mycoplasma contamination will show no cells, or a reduced number of cells. - The method can be quantitated by staining with methylene blue (see Appendix 5.3) or, as recommended by Gibco, by aspirating the medium and treating the cells at room temperature with 0.28 crystal violet in 10% formalin (buffered to pH 7.0 with 0.1 M sodium phosphate). After 20 min, remove the stain and wash the monolayer with distilled water. -

9.8.5. DNA probe for mycoplasma

Gen-Probe Inc. (Appendix 3) have brought out a kit to detect the presence of mycoplasmal ribosomal RNA in cell cultures. Although the first version was not very sensitive, and the second gave variable results (Johanssan and Bolske, 1989), mark I11 may be better but still costs about 10 times as much as the Hoescht test. The procedure involves: addition of a tritiated probe of mycoplasmal DNA to the cell lysate. - hybridisation at 72°C for 1 h. - precipitation and washing of the DNA/RNA hybrid. - counting of the precipitate for [3H]DNA. -

9.9. Elimination of mycoplasma Most contaminated cultures should be autoclaved to prevent spread of the mycoplasmas. Occasionally, a very important cell strain may become or be found to be contaminated, and it has been reported that kanamycin (0.1 mg/ml) prevents the growth (Fogh and Hacker, 1960) and tylocine (6.0 pg/ml) is also reported to be effective against PPLO (Gibco Biocult catalogue; Appendix 3). Boehringer

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Mannheim market a combination of two antibiotics for sequential use (BM cycle) and quinolones such as ciprofloxacin (Schmitt et al., 1988) and MRA (available from Flow Laboratories for use at 0.5-10 pg/ml) are very effective at eliminating mycoplasma. The quinolones inhibit the prokaryotic DNA gyrase. Mycoplasma can be eliminated from cell culture by treatment with immune serum (Pollock and Kenny, 1963) and passage through an animal is often effective in removing mycoplasma from tumour producing cell lines.

9.10. Viral contamination Since the possibility was recognised that bovine serum may be contaminated with several viruses (e.g. bovine herpesvirus and parainfluenza 3-virus) sera are now routinely tested by the manufacturer. The effectiveness of this screening has, however, been questioned (Kniazeff, 1973), and the use of serum-free media (0 5.8) is recommended as the only secure method of eliminating serum-borne viral contamination cytopathic effect. The problem is more serious, however, if no cytopathic effect is seen but the virus continues to replicate at a slow rate or in a few cells only. Thus primary cells from children with Burkitt’s lymphoma show no signs of Epstein-Barr virus (EBV) until several passages when a few cells show positive immunofluorescence (Henle and Henle, 1966). Cells transformed with the papovaviruses polyoma (e.g. BHK 21PyY, Dulbecco, 1968) and SV40 (e.g. SV28, Wiblin and Macpherson, 1972) show the presence of tumour antigen by immunofluorescence, but this is not harmful; it rather results in an increased growth potential and consequent disregard for neighbouring cells. Temperature-sensitivevariants of the BHK21 PyY cells have been selected, which at the non-permissive temperature are unable to make T-antigen, and the host cell reverts to its non-transformed character (Dulbecco, 1969). Thus the expression of viral functions depends on the conditions of culture, and often virulent virus may be rescued from non-permissive cells by fusion with uninfected permissive cells (Green, 1970). A similar in vitro rescue can be used

CH. 9.

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185

to detect the presence of cryptic virus in non-permissive cells. Thus a homogenate of the suspect cells is added to a permissive cell line or injected into a susceptible animal when the presence of a cytopathc effect indicates a positive response (Vigier, 1970). The mouse antibody (MAB) test involves injecting lo7 cells into prescreened mice which are bled out at 28 days and the sera tested for the presence of antibodies to a series of viral antigens. Such tests are mandatory if the cells are to be used for production of material for human treatment.

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CHAPTER 10

The cell cycle IO.I . Description Growing cells are characterised by a sequence or sequences of events leading to duplication of their constitutents. These events appear to occur in a strict, temporal order, and growing cells may be considered as a simple system for the study of gene expression. The two most obvious events which occur in growing cells are cell division and DNA synthesis which are the markers used to characterise the cell cycle (Fig. 10.1). M or mitosis is the period when the cells divide and S is the period of DNA synthesis while G1 and G2 represent gaps - gaps in our knowledge of obvious markers in these areas. Much cell biology in recent years has been devoted to attempts to fill these gaps. These

DNA

Gl

Fig. 10.1. The cell cycle. S is the DNA synthetic period. G1 and G2 are the gaps between Mitosis (M) and S, and S and M, respectively. 187

188

CELL CULTURE FOR BIOCHEMISTS

symbols (Gl, S, G2 and M) are used throughout the text to describe the phases of the cell cycle.

10.2. Mitosis The stages of mitosis are: prophase metaphase anaphase telophase Mitosis is heralded by the rounding up of the cell, and the first visible indication that it is about to divide is a change in appearance

Fig. 10.2. Mitotic stages. Mouse L929 cells, pulse labelled with tritiated thymidine, were processed for autoradiography. Among the S-phase cells (covered with grains) are cells in other stages of interphase and several mitotic cells. A metaphase cell, two anaphase cells and a late telophase cell are distinguishable.

CH. 10.

u=, oI

u

I

T H E CELL CYCLE

:;"

189

190

CELL CULTURE FOR BIOCHEMISTS

of the nucleus. This is caused by the condensation fo the chromosomes in early prophase, a process which continues as the nuclear membrane disappears, so that by metaphase the highly condensed chromosomes are massed in the centre of the rounded-up cell. At this stage the cells are only loosely attached to the substratum and are readily dislodged by agitation or by gentle trypsinisation (Fig. 10.2). This forms the basis of the method of selection synchrony of mitotic cells (see 5 11.2). The centrioles migrate to opposite poles of the cell and the mitotic spindle is formed, apparently joining the cell membrane through the centrioles to the centromere of each chromosome. Spindle fibres consist of one type of protein, tubulin, of molecular weight 60,000. It is the organisation of these molecules to form the mitotic spindle which is blocked by the drugs colchicine, colcemide, nocodazole, vincristine and vinblastine (Fig. 10.3) with the consequence that mitosis is arrested in metaphase. At anaphase the two sets of chromosomes move to opposite poles due to tubulin action, and by telophase the chromosomes decondense as the cell membrane encloses each new daughter cell. The paired cells are still rounded up and resemble a dumbell, but very soon they flatten out as the nuclear membrane and nucleoli reform and the cells enter G1.

10.3. S-phase S-phase is, by definition, the period in the cell cycle during which DNA is synthesised. It is clear, however, that DNA synthesis does not start suddenly, proceed at full speed, and then stop suddenly, and hence to define exactly the beginning and end of S-phase is virtually impossible. It has been shown using DNA fibre autoradiography (Cairns, 1966, 1972) that in animal cells the synthesis of DNA occurs in discrete, short stretches or replicons (Huberman and Riggs, 1968). Replicons vary in size from 15-60 pm but are mostly less than 30 pm long in tissue culture cells (but see Callan, 1972). DNA synthesis is initiated in the middle of a replicon and proceeds bidirectionally to the ends of the replicon (Hand and T a m , 1974). Later adjacent

CH. 10.

THE CELL CYCLE

191

replicons fuse and the two replicated chromatids can then separate (Kowalski and Cheevers, 1976). If cells are synchronised at the Gl/S boundary and then released, the rate of DNA synthesis is initially slow but accelerates to reach a maximum at about 3h and then decelerates until S-phase is essentially complete in 6-7 h (Stubblefield and Mueller, 1962; Adams, 1969b). As replication occurs different numbers of replicons are active at any one time, and so it is not surprising that more careful labelling reveals bursts of tritiated thymidine incorporation throughout S-phase rather than a steady even progression (Klevecz, 1969; Lett and Sun, 1970; Klevecz et al., 1974). This was shown clearly by Stubblefield and Mueller (1962), who demonstrated focalised synthesis of DNA by pulse-labelling a random population of cells with tritiated thymidine and then after varied time intervals visualised autoradiographically the regions of metaphase chromosomes where incorporation has occurred. One of the X-chromosomes in cells of female mammals has been found to replicate later than any other DNA in the cell (e.g. Gilbert et al., 1965), and particular satellites replicate at particular times in S-phase. Thus there is a specific temporal order in which particular replicons replicate. Detailed analysis using several different methods (Pulse and continuous label with tritiated thymidine, DNA fluorescence per cell using the fluorescent Feulgen assay and flow microfluorography (Van Dilla et al., 1969), and DNA specific fluorescence using the diaminobenzoic acid assay (Kissane and Robins, 1958) reveals that the DNA content of a cell increases in a saltatory fashion, and that the early portion of S-phase is a period of low net DNA synthesis which may be mistaken for G1 if insensitive methods of measurement are used (Klevecz et al., 1975).

10.4. Control of the cell cycle 10.4.1, The GO-phase and commitment to cycle

Although cells in culture and cells still in the body have similar durations for S, G2 and M, it is apparent that variations in the

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TABLE 10.1 Duration (h) of cell cycle phases in cultured cells Cell types

T

fG1

rs

fG2

HeLa Human fibroblasts Human amnion Mouse L Mouse L5178y Chinese hamster fibroblasts

20-28 16-30 19.4 18-23 11.5 12-15

8-16 3-16 9.8 6-11 1.5 3- 6

5- 9

2-8 4-5 2.2 3-4 2.9 2-3

6-11 6.7 6-12 7.1 4- 8

Results have been pooled from several sources (Firket, 1965; Cleaver, 1967; Lipkin, 1971; Puck, 1972) to give a general range of data which should be compared to the results for human amnion cells (Sisken and Morasca, 1965) and mouse L5178Y cells (Defendi and Manson, 1963). As the duration of mitosis is short and not always reported, where known it has been shared between fG1 and fG2. T is the total cell generation time; rG1, rS and rG2 are the durations of the G1, S and G2-phases, respectively. (See Fig. 10.1 for a diagrammatic representation of the cell cycle.)

duration of G1 can be dramatic and in fact account for the major variation in cell cycle time of different cell types (Table 10.1). Thus G1 is apparently absent in cultured Chinese hamster lung cells (Robbins and Scharff, 1967) and no upper limit has yet been placed on its duration. When Chinese hamster cells lacking a G1-phase are fused with Gl+-cells (5 13.5) the G1- state is dominant (Liskay and Prescott, 1978). Cells whch have spent a long time in G1 lose some of the enzymes typically present in dividing cells - particularly those concerned with DNA synthesis. These cells have traditionally been said to be ‘out of cycle’ or in GO. The implication of the GO-phase is that to leave GO-cells require a stimulus to urge them past a barrier and back into cycle. Pardee (1974) has suggested that whenever cells are exposed to suboptimal physiological conditions they enter a quiescent phase, and that there is a single restriction point in G1 which regulates their re-entry into a new round of the cell cycle. There are many experimental conditions where cells are put into to or taken out of GO in order to study the changes associated with onset of proliferation. The following examples are referred to elsewhere in this book.

CH. 10.

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193

1) Lymphocytes may be stimulated to grow by mitogens (0 6.2). 2) Serum starved cells may be stimulated by readdition of serum (Burke, 1970 and 0 11.6). 3) 3T3 cells may be stimulated to grow by treatment with E G F and/or prostaglandin PGF,, (de Asua et al., 1981 and 6 2.5 and 11.6). 4) Cells which have ceased to grow exponentially and reached a plateau phase because of limitations of growth surface may be stimulated by subculturing (Stoker, 1972 and 0 11.5). However, even in these non-growing cell populations a small number of cells are found to be making DNA or dividing, and it has been suggested that although most of the cells are in GO, a small proportion has been stimulated into proliferation. The question is: does the chance of a cell leaving GO and entering into cycle depend on the length of time since it last divided (the deterministic model), or is there an equal probability for any cell to enter the cycle (the probabalistic model)? This latter alternative has been expressed most forcibly by Smith and Martin (1973, 1974). They have named S-phase, G2,M and part of the G1 the B-phase, and suggest that the duration of the B-phase is fixed within narrow limits for a particular cell type. Shortly after mitosis cells enter the A-state in which the cells do not progress towards division. A cell may remain in the A-state for any length of time but always has a fixed probability ( P ) of leaving for the B-phase, provided environmental conditions remain constant. Smith and Martin suggest that modification of the transition probability provides a major means of controlling cell proliferation The two models of cell proliferation differ largely in the prediciton of the Smith and Martin model that the proportion of cells remaining in interphase (a) should decline exponentially with age, beginning at time TB after mitosis. Thus a semilog plot of a against age after mitosis should show a linear decay after a lag period equal toTB. As shown in Fig. 10.4 this is borne out by the data. In fact there is an initial downward curvature before linearity is attained caused by the variability in TB.

194

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1

"

'

! 10

20

,

,

, 30

,

, 40

,

, 50

Age ( h )

Fig. 10.4. Distribution of generation times of various cell types in culture. The proportion (a)of cells remaining in interphase at various times after division has been recorded for various cells by time lapse cinematography. (a) Rat sarcoma, (b) HeLa S3, (c) mouse fibroblasts (d) L5 cells and (e) HeLa. (Reproduced from Smith and Martin, 1973, with kind permission of the authors and publishers.)

The alternative model would predict that the greater the age of a cell the greater is its probability of division and hence the semilog plot of a! against age would constantly curve downwards. In practice it is exceedingly difficult to distinguish the two possibilities and there is considerable discussion as to which model is correct (Baserga, 1978). The traditional model of the cell cycle explains differences in generation times between the cells in a population as a result of the sum of small differences in the times taken for the large numbers of steps required for a cell to progress from one division to the next. In the model of Smith and Martin (1973), although such differences do occur and will lead to the heterogeneity in the length of the B-phase, the major contribution to the variation in generation time between cells is the length of time spent in the A-state. Minor and Smith (1974) argue that variations in the duration of the B-phase for sibling pairs should be minimal, and they find a strong correlation between intermitotic times of siblings and the overall variability of the population.

CH. 10.

THE CELL CYCLE

195

However, Pardee (Pardee et al., 1979) argues that the a-plots of Smith et al. (e.g. Fig. 10.4) overemphasize the lagging cells and disguise short cycle-time cells. Small and large cells from a quiescent culture have been sorted (using the fluorescence activated cell sorter, 6 10.7.5) and their cycle times measured (Shields et al., 1978). Small cells have a longer period before division but all cells divide as normal sized cells; i.e. there is no selection for rapidly growing cells. The distinction between parental cells is lost in the daughters, showing that heterogeneity is environmentally produced and this distinction can be explained equally well by deterministic or proabilistic models. 10.4.2. p34 and cyclins

A 34 kDa protein (p34) plays an important function in the control of

the cell cycle in all eukaryotes. It was first identified as the product of the cdc 2/cdc 28 gene in yeast mutants which caused cells to be arrested at a ‘commitment point’ in G1 (Murray, 1981). However, anti-p34 antibodies injected into cells do not affect DNA synthesis but block cells in mitosis (Riabowol et al., 1989) and p34 is believed to function both at the onset of S-phase and at mitosis. Cyclins are proteins which vary dramatically in abundance during the cell cycle. Cyclin M is a 62 kDa protein (the homologue of the 65 kDa product of the yeast cdc 13 gene) which accumulates during S phase in HeLa cells and binds to a p34 which is phosphorylated on tyrosine 15 (Draetta and Beach, 1988; Gautier et al., 1989; Morla et al., 1989; Gould and Nurse, 1989; Murray, 1989; Dunphy and Newport, 1989). This leads indirectly to dephosphorylation of p34, which is thereby activated to become a serine/threonine protein kmase which autophosphorylates cyclin M. Later p34 also becomes autophosphorylated at serine/threonine which leads to dissociation of cyclin M and formation of a so-called ‘maturation promoting factor’ (MPF) which retains the protein kinase activity. The tyrosine dephosphorylation of p34 and phosphorylation of cyclin M precipitates cells into mitosis, while the formation of active MPF leads to phosphorylation of histone H1 and the completion of, and exit from, mitosis.

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Another substrate for the cyclin M/p34 protein kinase may be the carboxy terminal region of the large subunit of RNA polymerase I1 and this may be related to the switch off of transcription during mitosis (Cisek and Corden, 1989). MPF is inactivated (probably by dephosphorylation) following interaction with a 13 kDa polypeptide (homologous to the product of the yeast suc 1 gene). Cyclin G1 appears in G1 and is degraded as cells enter S-phase. It interacts with the p34/p13 complex, releasing pl3, and reactivating the serine/threonine kinase activity, one substrate of which is cyclin G1. This leads to initiation of S-phase, though the reactions involved here are unknown (Lee and Nurse, 1988; Murray, 1989). As cyclin G1 is degraded, p34 becomes phosphorylated at tyrosine 15 and can again interact with the accumulating cyclin M. This scheme is still not completely substantiated but explains how the alternation of S-phase and mitosis can be achieved. It is based on the protein kinase activity of p34 which is active under three sets of condition: 1) unphosphorylated but bound to phosphorylated cyclin M. 2) unphosphorylated but bound to phosphorylated cyclin G1. 3) in a phosphorylated form as MPF. The substrates of the three forms of p34 protein kinase are different.

10.5. Distribution of cells around the cycle For every cell which enters mitosis at the end of the cell cycle two will begin the next cycle. This means that the distribution of cells around the cycle is not uniform, but that there is a preponderance of young cells during exponential growth. Thus the ideal distribution of cells around the cycle is shown by the solid line in Fig. 10.5 (Engleberg, 1961; Kubitschek, 1966). In practical terms because of the variability in generation times the dotted line in Fig. 10.5 more accurately reflects the real situation (Sisken and Morasca, 1965).

C H . 10.

THE CELL CYCLE

197

Cell number1

Fig. 10.5. Distribution of exponentially growing cells around the cell cycle. Cells in an exponentially growing population are theoretically distributed round the cell cycle as shown by the solid line. However, as the cell cycle time varies amongst the cells the dotted line more closely resembles the observed distribution. Cells which have just divided have an age of zero while those in the next mitosis have age T. The positions of G1, S and G2 are shown for a typical cell. (Reproduced from Cleaver, 1967, with lund permission of the author.)

If the cells do not all have the same generation time then the time taken to double the number of cells (T,,: the cell doubling time) will be slightly shorter than the generation time ( T ) .Alternatively, if not all the cells in the population are growing i.e. the growth fraction is less than 1 (see below) then TD will be longer than T. As well as there being twice as many cells at the beginning of G1 as at the end of G2, similar but smaller differences will exist between cells at the beginning and end of S-phase. Moreover, the proportion of cells in S-phase not only depends on the relative lengths of S-phase and the generation time but also on the location of S-phase within the cycle (but see ‘growth fraction’ below). It is, therefore, not only inaccurate to say that, if 30% of cells are in S-phase (labelling index LI = 0.3) then the duration of S-phase ( t S ) is 30% of the generation time ( T ) ,but it is even inaccurate to say that LI=ln2.-

tS T

(10.1)

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CELL CULTURE FOR BIOCHEMISTS

Rather, the location of S within the cycle must be defined and this is most easily done by relating it to T and tG2 to give

T

(10.2)

(Cleaver, 1965). Because the location of mitosis is fixed and its duration short, one can, however, say that the proportion of cells in mitosis, the mitotic index,

MI

= In

tM 2. T

(10.3)

(Smith and Dendy, 1962). However, a generalisation is that, while G1-cells have a single complement of DNA and G2-cells have twice this amount, expontentially growing cells have an average of about 1.3 times as much DNA as does a G1-cell.

10.6. Growth fraction In most cell populations not all the cells are proliferating, there being a proportion of non-proliferating cells. These may alternatively be described as being in GO or in the A-state. The proliferation index of growth fraction is given by N, N

-=

number of proliferating cells total number of cells

(10.4)

The time taken for the total number of cells to double (cell doubling time) is therefore not equal to the cell cycle or cell generation time. It is important, therefore, when doing cell cycle analyses to carefully distinguish between T and TD and preferably maintain the growth fraction as high as possible. Low growth fractions are common in vivo (even some tumours may have a growth fraction less than O.l), and very often in vitro in

CH. 10.

199

THE CELL CYCLE

Fig. 10.6. Distribution of steady state cells around the cell cycle. In the steady state the number of cells in a particular phase is proportional to the duration of that phase.

primary cultures or under suboptimal conditions not all the cells are growing. The low growth fraction may arise by irreversible differentiation by cells entering GO or A-state by cell death. In the steady state, additions to the population are balanced by subtractions from it and the age distribution is rectangular (Fig. 10.6), i.e. the fraction of cells in a phase is now directly proportional to the duration of that phase e.g. Mitotic index (MI)

=

tM

T

(10.5)

Further information on cell cycle kinetics can be found in Cleaver (1967) and in Aherne et al. (1977).

10.7. Cell cycle analysis 10.7.1. Tritiated thymidine pulse method (Howard and Pelc, 1953)

This is perhaps the earliest method used, and the following description is for cells growing in small dishes or on coverslips. The method,

200

CELL CULTURE FOR BIOCHEMISTS

however, can be modified for use with suspension cultures. Set up a series of coverslips in multiwell dishes (see 0 2.2). 48 or more coverslips are required, i.e. 2 multiwell trays. Each well should be seeded with 1-2 X l o 4 cells in 0.5 ml medium and the cells allowed to incubate overnight to ensure exponential growth. It is preferable to use Hepes buffered medium as the regular harvesting required makes it very difficult to maintain pH with a bicarbonate buffer. It is also important not to let the temperature fall during sampling which should be done in a 37°C room. - Pulse label each culture for 30 min with [3H]thymidine (5 pCi/ml; 2 Ci/mmol), i.e. to each well containing 0.5 ml growth medium add 10 pl of a solution containing 2.5 pCi [3H] thymidine at a concentration of 1.25 X l o 4 M. - Remove the [3H]thymidine containing medium using an unplugged Pasteur pipette connected to a vacuum pump through a trap. Quickly replace with prewarmed medium containing 2 X M thymidine. - Fix coverslips at frequent intervals (20-30 min) by dipping the coverslip into PBS (twice); 5% cold trichloroacetic acid (4 times) and absolute ethanol (twice). Attach the coverslips to one end of a glass slide (cells uppermost) using DePeX (Pearse, 1953) and process for autoradiography (see 6 12.4). DePeX is a mountant available from G.T. Gurr Ltd. (Appendix 3). - Record the proportion of mitotic cells labelled. In theory the first labelled mitotic cell will appear after a time equal to the length of G2 (tG2) and the percentage of labelled mitotic cells should rise to 100 over tM (Fig. 10.7). After a further tS the percentage of labelled mitotic figures should fall and will only rise again after tG1+ tG2. -

In practice, because of the variability between cells and difficulties in producing short pulses all boundaries are blurred. However, by taking 50% values for the various transitions a reasonable approximation to the duration of the cell cycle phases can be obtained.

CH. 10.

201

THE CELL CYCLE

M

L

h

Fig. 10.7. Fraction of labelled mitoses. The solid line represents the theoretical change in the fraction of mitotic cells after labelling for 100 min with tritiated thymidine. The dotted line follows data obtained with mouse L cells. The hatched areas represent the duration of the exposure to tritiated thymidine. (Reproduced from Cleaver, 1967, with kind permission of the author.)

10.7.2. Continuous labelling method -

The cells should be set up and labelled as before except that

- [3H]thymidine is used at 2.5 pCi/ml(0.06 Ci/mmol). Do not remove the growth medium with the tritium labelled thymidine but continue to incubate and harvest coverslips. - Process for autoradiography and record the fraction of labelled mitoses, the fraction of labelled cells, and the grain count, i.e. the average number of autoradiographic grains over the labelled cells (Fig. 10.8.).

-

The fraction of cells labelled will reach 1.0 only when all the cells not initially in S-phase have proceeded around the cycle and entered S-phase, i.e. after T-tS which in this case = 16.3 h = tG1 + tG2 tM.

+

202

CELL CULTURE CULTURE FOR FOR BIOCHEMISTS BIOCHEMISTS CELL

Labelled interphase cells 02

I

Fig. 10.8. Continuous labelling method. Human skin epithelial cell cultures were exposed to tritiated thymidine (2.5 pCi/ml; 0.06 Ci/mol) continuously for 25 h during which time cells were harvested and scored for the indicated parameters. (Reproduced from Cleaver, 1967, with kind permission of the author.)

tG2 can be found as before by the time taken for the first labelled mitosis to appear. Thus tG1 can be found by difference. The grain count will plateau when the labelled cells have progressed through the whole of S-phase in the presence of [ 3H]thymidine. However, when these cells divide the grains are now shared between the two cells and the grain count will fall. This introduces errors into the estimated t S . Errors also are introduced by the presence of those cells already in S-phase when the thymidine is added or when the cells are harvested. This method therefore overestimates the duration of t S .

CH. 10.

203

THE CELL CYCLE

10.7.3. Accumulation functions

A certain amount of information can be gained by measuring the accumulation of mitotic cells on addition of a mitotic blocking agent. Cultures may be set up as before and colcemid (0.25 pg/ml) added at zero time. Figure 10.9 shows that after a short lag the plot of the accumulation function (log (1 N m ) where Nm = fraction of mitotic cells) against time is linear. From such a curve the generation time ( T ) can be calculated either (a) from the equation

+

log(1 + N , )

=

0‘301 ( t M + t ) T

(10.6)

where tM is the time spent in mitosis and t is the time from addition of colcemid, or (b) by measuring the time required for the accumulation function to reach 0.3. In practice this seldom happens in the theoretical manner as (1) the generation time differs from cell to cell, ( 2 ) 100% of the cells may not be viable, (3) some cells may escape the colcemid block and re-enter G1, and (4) colcemid may have

o,06LP

0.02

I-’, 2 4

I

I

I

I

6

8

10

,

,

12

14

,

16

,

,

18 20

,

22

h

Fig. 10.9. Accumulation of mitotic cells. The mitotic collection function (log 1 + N m ) is plotted against time for Chinese hamster cells (T=12.4 h) and HeLa S3 cells ( T = 20.1 h) to whch colcemid was added at zero time. (Reproduced from Puck, 1964, with kind permission of the author and publisher.)

204

CELL CULTURE FOR BIOCHEMISTS

deleterious effects on other parts of the cell cycle thus affecting the generation time. The lag in the rate of accumulation of mitotic cells immediately following addition of colcemid is explained by the fact that cells already in mitosis on addition of the drug are not blocked but complete mitosis and re-enter G1.Taylor (1965) has shown that with the related drug colchicine this happens at concentrations between 60 and 200 nM, but at concentrations above 200 nM all cells are blocked in mitosis. If tritiated thymidine is added along with colcemid three accumulation functions can be measured: (a) total labelled cells, (b) total mitoses, (c) labelled mitoses, which enables calculation of the length of S and G 2 and hence also G1. Thus from Fig. 10.10 it can be seen that an accurate measure of tG2 can be obtained from the distance between the lines which should be parallel if the cell population is exponential and if the tritiated thymidine is not having any deleterious effects on growth.

0.300.26-

labelled m Itoses

2

6

10

18

14

22

26

h

Fig. 10.10. Accumulation of labelled mitotic cells. A similar experiment with HeLa S3 cells to that shown in Fig. 10.9 except that ['Hlthymidine was added with the colcemid at zero time. After an initial lag the lines for accumulation of all mitotic cells and labelled mitotic cells are parallel and separated by rG2. (Reproduced from Puck and Steffen, 1963, with kind permission of the authors and publisher.)

CH. 10

205

THE CELL CYCLE

0.12

0.10

0.08 0.061

;

I

3

5

I

7

I

9

I

11

11 ‘I

I

24

Fig. 10.11. Accumulation of labelled cells. The data points for this experiment can be obtained from the experiment described in Fig. 10.10. See text for details. (Reproduced from Puck and Steffen, 1963, with kind permission of the authors and publisher.)

The generation time ( T ) can be obtained from the slope of the line which equals 0.3/T. Using the figure for tG2 so obtained a second graph may be drawn (Fig. 10.11) of time against

log( 1

+

q)

(10.7)

where N ( L ) is the fraction of cells labelled and

(

k = e x p In 2 * r;2)

(10.8)

(Puck and Steffen, 1963; Puck et al., 1964). This function reaches a plateau when all the cells in G1 have entered S-phase (or, if an intermediate concentration of colchicine is used, all the M + G1-cells have entered S). As there is signficant variation in the lengths of G1

206

CELL CULTURE FOR BIOCHEMISTS

among the cell population this is not a sharp cut off, but the line obtained from the points over the first 6 h or so can be extended, and all that is required is a final figure which can be obtained at 24 h, the intercept 0.3 tS/T gives a value for t S . Although this method involves a more difficult mathematical background [for which the original references or Cleaver (1967) should be consulted] it involves few time points spread over a shorter period. 10.7.4. Graphical analysis

Perhaps the simplest method of cell cycle analysis is that described by Okada (1967) which gives the additional information of the proportion of cells in each phase of the cycle. The information required from an exponentially growing culture of cells is: a) the generation time which may be obtained as the cell doubling time or as an accumulation function as described above (see 8 10.7.3) b) the mitotic index (see 0 10.5) c) the percent of cells in S-phase obtained by pulse labelling with [ 'Hlthymidine (see 6 10.7.1) d) the duration of G 2 obtained either from the time after addition of [3H]thymidine to the appearance of a labelled mitotic cell or from the separation between the lines in Fig. 10.10. All this information may be obtained in an experiment lasting 3-4 h with or without the use of colcemid. The graph (Fig. 10.12)is then plotted on single cycle semi-log paper as follows: - Mark off the generation time on the linear axis and divide the region from 1 to 2 on the log axis into 100 divisions (percentages), putting 0% at 1 and 100% at 2. Draw a straight joining 100%to T. - From T draw a vertical line a length corresponding to the percentage of cells in mitosis and join this to the diagonal line to give the duration of M . - Continue the horizontal line for tG2 and join this to the diagonal line to give the percentage of cells in G2.

CH. 10.

THE CELL CYCLE

207

h

Fig. 10.12. Graphical analysis of cell cycle. 5 cm Petri dish cultures of Aedes albopictus mosquito cells were set up and the number of cells per dish counted regularly to establish the doubling time (taken as the generation time). When it was clear that the cells were growing exponentially ['Hlthymidine and colcemid were added and cells processed to establish % cells labelled, mitotic index and rG2. The graph was drawn as described in the text (see 5 10.7.4.)

-

Draw a vertical line corresponding to the percentage of cells in S and join this to the diagonal to give tS.

The final horizontal and vertical lines give tG1 and the percentage of cells in G1. 10.7.5. Flow rnicrojluorornetry (fluorescence activated cell sorting,

FA CS)

An instrument developed at the Los Alamos Scientific Laboratory permits analysis of large numbers of single cells with respect to DNA and protein content, cell volume, etc. (Fulwyler, 1965; Kraemer et al., 1973; Klevecz et al., 1975; Herzenberg et al., 1976). The output data from a large population of cells consists of, for example, a distribution of the values of cellular DNA content or cell volume. In addition to the capability for complex analyses the instrument is able to physically separate particular cell subpopulations of interest. Cells are stained with fluorescent dyes specific for a particular cell constituent (e.g. ethidium bromide at 0.1 mg/ml stains DNA in

208

CELL CULTURE FOR BIOCHEMISTS

isolated nuclei (Vindelor et al., 1983) or can be used to identify dead cells), or a series of antibodies each conjugated to a different fluorochrome may be used. Modern machines can process and display in 8-dimensions from a single pass. The stained cells are analysed as they pass in single file across sensing devices. Thus during transit (2-3 psec) across an argon ion laser beam a fluorescent light flash is emitted from each cell which can be analysed for both intensity and colour. In addition cell volume and cell granularity signals may be generated by measuring light scattered forwards and at right angles to the incident beam. Cell sorting is achieved by first breaking the stream of cells into droplets such that 1%of the droplets contain a single cell. Droplets are formed and pass between electrically charged plates a fixed time after analysis. Cells of particular characteristics (e.g. containing a G2 amount of DNA) can be sorted by giving the cell stream a charge for a short time such that three droplets (the centre one of which is calculated to carry the cell of interest) are deflected from the main stream into a separate collecting tube as they pass between the charged plates. To give some idea of the results CHO cells pulse labelled with [ 'Hlthymidine were stained with either ethidium bromide or using the acriflavin-Feulgen method (again staining DNA) and submitted to flow microfluorometry and cell sorting. Figure 10.13 shows the distribution of DNA content per cell. Approximately 61% of the cells had the G1 content of DNA and about 16.3% had the G2 + M content. The remaining 23% of the cells fell in between these two values. Use is made of the formulae derived by Lennartz and Maurer (1964):

= the fraction of cells in the population which occupy the interval dB of the cell cycle at any one time t . It is this formula which gives Fig. 10.5 and from which values for tG1 (8.4 h), t(G2 M) (3.5 h) and t S (4.1 h) may be obtained.

+

CH. 10

THE CELL CYCLE

Sort 1

Sort 2

209

Sort 3

Channel n u m b e r

Fig. 10.13. Distribution of cells separated by flow microfluorometry. CHO cells were pulse labelled for 15 min with [3H]thymidine (1 FCi/ml) and stained with ethidium bromide. They were then submitted to flow microfluorometry and cell sorting on the basis of cellular DNA content. Cells from the indicated portions (sort I, 2 and 3) were then subjected to autoradiography and shown to contain respectively 4%.93% and 19% of the cells labelled. (Reproduced from Kraemer et al., 1973, with kind permission of the authors and publisher.)

The leading edge of the G1 peak of cells was shown by autoradiography to have only 4% of the cells labelled and similarly the trailing edge of the G2 + M population had 19%of the cells labelled. Cells sorted from between the two peaks had 93% labelled. The sensitivity of the method is shown by the detection of a minor peak representing less than 1%of the cell population with a DNA content twice that of G2 + M cells. A computer program was developed (Dean and Jett, 1974) to resolve the flow microfluorometric distribution into G1, S and G2 M subpopulations and enable cell cycle analyses to be performed rapidly and accurately. This has been constantly improved and is incorporated on the software of modern FACS machines (e.g. the Becton Dickinson FACScan) (see 6 11.4.3 and Fig. 11.3). Some

+

210

CELL CULTURE FOR BIOCHEMISTS

of the problems with data analysis have been reviewed by Dean (1987). Instead of labelling with [ 3H]thymidine(which requires collection of cells and their processing for autoradiography), the cells may be pulse labelled with bromodeoxyuridine prior to analysis. They are then lysed and treated with propidium iodide (to give a fluorescence signal proportional to the amount of DNA per cell) and an FITCconjugated antiBudr antibody (6 12.3) which will indicate those cells making DNA at the time of harvest. It is clear from the dual analysis that those cells with intermediate amounts of DNA (Le. S-phase cells) are those which react with the antiBudr antibody. Cell sorting takes place at a rate of 300,000 cells/min and for this reason a FACS machine is more usually used as an analytical tool rather than in a preparative mode. In addition to its use in cell cycle analysis, it can be used (a) to analyse the distribution of lymphocytes carrying a series of different surface antigens (e.g. to determine the proportion of T4 lymphocytes in a blood sample); (b) to estimate the proportion of dead cells in a population (i.e. cells which stain with propidium iodide without prior fixation); or (c) to determine the proportion of transformed cells (i.e. cells bearing a particular surface antigen) in a culture or biopsy (Watson, 1987).

CHAPTER 11

Cell synchronisation

11.1. Introduction Two different principles are used when a population of cells at a unique position in the cell cycle is required. a) It is possible to block cells so that they accumulate at a specific stage in the cycle. This may be done chemically or physiologically but such methods suffer from the disadvantage that the cells have been interfered with and are therefore already abnormal in certain respects (see, however, 0 11.7). b) Cells at a particular stage of the cell cycle may be selected. This may be on the basis of some physical property, e.g. the loose attachment of mitotic cells or the varying size or DNA content of cells at different stages of the cycle. Alternatively, it may involve a chemical characteristic allowing remaining cells to be selectively killed. Which method is chosen depends on the requirements of the experiment. It is sometimes preferable to perform the initial stages of an experiment (e.g. isotopic labelling) with an unsynchronised exponentially growing culture and then to select cells at a given phase of the cycle by e.g. zone centrifugation for enzymic analysis. In such experiments the cells, following selection, are not generally replaced in culture and so they may be exposed to an otherwise deleterious environment. Several different methods of synchronising CHO cells have been compared by Grdina et al. (1987). 211

212

CELL CULTURE FOR BIOCHEMISTS

11.2. Selection of mitotic cells Cells growing as a monolayer round up when they divide. They therefore are less firmly attached to the substratum at this time and may be easily detached (Terasima and Tolmach, 1961, 1963; Robbins and Marcus, 1964; Peterson et al., 1968; Shall, 1973a). Exponentially growing cells are used and it is important to maintain constant pH and temperature throughout the selection procedure. Klevecz et al. (1974, 1975) have used CHO cells growing in McCoy’s medium with 20% foetal bovine serum and Hepes buffer. They maintain the cells throughout in this medium at 37°C and, of the cells they select, 98-99% are in mitosis. The viability of these selected cells approaches 100% and on reseeding half the cells attach within 1 h of selection and maximum attachment is found by 4 h (Klevecz, 1975). However, they select a very small proportion of the original cells. As different cells attach with differing firmness to the substratum, the following procedures may need to be modified for each cell type. 11.2.1. Shaking

Inoculate cells into bottles 24 h prior to initial selection. 5 x lo7 Don-C Chinese hamster fibroblast cells may be inoculated into a roller bottle in McCoy’s medium (see 6 11.2.2). - After 24 h shake the bottle of cells (at least lo7 cells should be present initially) and discard the medium which contains dead cells and cell debris. The shaking is best done in a shaking water bath so that the bottle is shaken 20 times in 3 sec with the medium washing over the cell monolayer. The firmness of attachment of different cell lines differs considerably and the vigour required to dislodge mitotic cells must therefore be investigated anew for each case. - Add fresh prewarmed medium (take care not to pipette it onto the cell monolayer) and reincubate. - After periods of 15, 30 or 45 min (the actual interval varies with cell line), harvest loosely attached cells by shaking as above and this time collect the medium on ice, pooling all batches. -

CH. 11.

-

-

CELL SYNCHRONISATION

213

Sediment the cells at 700g for 3 min. Reseed the mitotic cells at about lo5 per 5 cm dish, when they should attach and divide within an hour.

Although the yield of mitotic cells is poor (1-3%) it is increased by pooling many harvests taken over a 2-3-h period. Another way of increasing the yield of mitotic cells is to accumulate cells in metaphase by a brief treatment with colcemid or nocodazole. By subculturing Don-C Chinese hamster fibroblasts over 24 h Stubblefield et al. (1967) achieved a partial synchronisation with mitotic peaks at 7 and 19 h after subculture. By adding colcemid at 18 h and selecting mitotic cells at 22 h after subculture they obtained a yield of 21%of the population, 86%of which were in metaphase. These cells grew normally and exhibited no significant deviations from control cultures in their mitotic interval, generation time, DNA synthesis kinetics or proliferative capacity. This method, however, is not universally applicable as not all cell lines show reversible colcemid inhibition. Nocodazole has an advantage over colcemid in that cells do not escape the block and treatment for 4-5 h has no deleterious effect on cells (Zieve et al., 1980). 11.2.2. Trypsinisation

Stubblefield et al. (1967) used dilute trypsin to release their accumulated metaphase cells and claim that the purity of the resulting suspension was better than with shaking. The details of their method are: Subculture Don-C cells every 24 h in McCoy’s 5a medium (Appendix 1)containing 20%foetal calf serum and 0.08%lactalbumin hydrolysate, seeding cells at 1.2 x 1O5/d(3 x l o 4 cells/cm2). - 18 h after subculture add colcemid (0.06pg/ml) and continue incubation for 4 h. Some mitotic cells float free at this stage and should be combined with those released later by trypsinisation. - Remove the medium and replace with 0.1% cold trypsin in PBS. - Shake the cultures gently (about 2 cycles/sec) either by hand or in a shaking water bath.

-

214

-

-

CELL CULTURE FOR BIOCHEMISTS

After 45 sec transfer the suspended mitotic cells to centrifuge tubes, pellet at 400g for 2 min, and resuspend in fresh medium at 4 x lo4 cells/ml. After 20-30 min the cells divide synchronously. Again this method is not universally applicable as not all cell lines show reversible colcemid inhibition.

11.2.3. Selection from microcarriers

When cells are growing on microcarriers (9 3.6) mitotic cells can be selectively dislodged by increasing the stirring speed (Crespi and Thilly, 1982). -

-

Set up a microcamer culture containing 0.5 g microcarriers (e.g. Flow superbeads) and 3 X lo7 to 10' cells in 100 ml growth medium. Stir at 60 r.p.m. for 20 h. Increase the rate of stirring to 150 r.p.m. for 10 min to detach dead cells. Wash twice with prewarmed medium. Reincubate in the presence of colcemid at 30 ng/ml for 2.5 h, stirring at 60 r.p.m.. k n s e three times in prewarmed medium. Stir at 150 r.p.m. for 10 min. Allow microcarriers to settle under gravity and decant the supernatant through a 50pm nylon screen. Make the suspension lo5 cells/ml and plate 15 ml into 100 mm dishes.

In order to produce a stirring rate of 150 r.p.m. Crespi and Thilly (1982) made a special harvesting vessel with stirring bars. Their selected cells had a viability of 80-95% and a mitotic index of 85-95%. This approach is unlikely to be successful with the glass or plastic beads to which cells appear to attach only poorly at all stages of the cell cycle.

CH. 11.

CELL SYNCHRONISATION

215

11.3. Selective killing of cells in particular phases This is usually done either with high specific activity tritiated thymidine (Whitemore and Gulyas, 1966) a method which is of fairly general applicability or with hydroxyurea (Sinclair, 1965), a method applicable only to certain susceptible cells. The aim is to kill cells in a particular phase of the cell cycle, thereby selecting for growth those cells outwith that phase. As the cells are growing exponentially and passing round the cell cycle, the longer the period of exposure the fewer survivors and the narrower the width of the surviving population. This may be true in theory, but in practice if the killing is allowed to remove 90% of the cells, those remaining rather than being concentrated just before S-phase have in fact selected themselves as slow growers, and it may take several hours before they enter S-phase, and by the time they enter G2 most synchrony has been lost. Moreover, a major disadvantage with such methods is that the dead cells, although unable to divide, may continue to metabolise for a considerable period, thus confusing any biochemical investigation. Cells in mitosis may be selectively removed and discarded over a long period of time. This leaves behind a population most of which should be in G2-phase (Stubblefield, 1964; Creasey and Markiw, 1965; Pfeiffer and Tolmach, 1967). Unfortunately, as well as the probability of selecting slow growers, the use of colchicine, colcemid and vinblastine may not only inhibit mitosis but may also have other effects on cells such as inhibition of RNA synthesis.

11.4. Selection of cells by size 11.4.1. Electronic cell sorting

This is considered in more detail in fj 10.7.5. It is a method which involves a modified electronic cell counter, i.e. a Coulter counter linked to a pulse height analyser and an electronic cell sorter. It is capable of separating cells at a rate of about 50,00O/min into a

216

CELL CULTURE FOR BIOCHEMISTS

number of size classes. The cells remain viable and show no change in generation time (Fulwyler, 1965; Van Dilla et al., 1967). However, because of the high cost of the instrumentation and the relatively slow sorting rate this method has found limited applications in biochemistry at present. As an alternative to the use of the electronic cell counter, cell size may be analysed by measuring scattering of light from a laser (9 10.7.5.). 11.4.2. Zone sedimentation

The rate at which cells sediment depends on their density and size. It is therefore relatively easy to separate cells of different types which differ in density but more difficult to separate cells of the same type (but at different stages of the cell cycle), as in this case the rate of sedimentation (mm/h) is roughly equal to r2/4 where r is the cell radius in pm. A 2-fold increase in volume (the maximum to be expected) results in only a 1.6-fold increase in sedimentation rate. In order to stabilise the sedimenting cells, it is necessary to include a gradient and those most commonly used are serum or Ficoll (Pharmacia Ltd.) (Boone et al., 1968; Miller and Phillips, 1969; Macdonald and Miller, 1970; Warmsley and Pasternak, 1970). Sucrose has also been used (Sinclair and Bishop, 1965; Morns et al., 1967; Shall and McClelland, 1971; Shall, 1973b) but tends to lower cell viability, unless great care is taken to maintain isotonicity. Boone et al. (1968) centrifuge cells through a discontinuous 10-20% Ficoll gradient made up in Eagle's minimum essential medium modified for suspension (i.e. lacking calcium and bicarbonate and containing 10 times the normal phosphate concentration). They use an A-1X zonal centrifuge rotor and spin for 1 h at 1000 r.p.m. at 20°C, and obtained clear separation of different cell types (HeLa and rabbit thymocytes). Pretlow et al. (1978) showed that a shallow gradient of Ficoll (2.7-5.5%) centrifuged at low speed (about 1OOOg) for about 100 min, allowed clear, isokinetic separation of spheres differing 2-fold in volume. The density change in the gradient was small (1.017-1.027 g/ml) and viscosity effects negligible.

CH. 1 1 .

211

CELL SYNCHRONISATION

Sinclair and Bishop (1965) and Morris et al. (1967) also use a centrifuge to increase the rate of sedimentation, but Shall (1973b) and Miller and Phillips (1969) use sedimentation under unit gravity and both describe simple pieces of apparatus for construction of the gradients and obtaining the separation. Although Shall claims up to lo9 cells may be separated at a time, the maximum that can be separated on the Miller and Phillips apparatus is 3 X 10'. The two pieces of apparatus are essentially the same and are shown diagrammatically in Fig. 11.1. If made of glass the apparatus may be sterilised by autoclaving (cover the open ends of the tubes and reservoirs with aluminium foil) and the cells used for further growth. The gradient is formed from two solutions. A, contains either (a) 10% w/v sucrose in complete medium which has the NaCl concentration reduced by 146 mM to maintain constant osmotic pressure, or (b) 30% foetal calf serum in PBS; A, contains either (a) 2.72% w/v sucrose in compelte medium which has the NaCl concentration reduced by 40 mM or (b) 15% foetal calf serum in PBS. The volumes, and hence heights, of the two solutions must be the same (about 250 ml).

I ,

C

..

I

"rn

I

I X

w

I

10 c m

Fig. 11.1. Apparatus for separating cells by sedimentation under unit gravity (see text for description).

218

-

-

-

-

-

CELL CULTURE FOR BIOCHEMISTS

With tap X closed, open W and turn the bar magnet using a magnetic stirrer. The magnet should rotate rapidly to ensure thorough mixing of the components. Into B place 10-30 ml of (a) serum-free medium or (b) PBS and allow it to run into C to remove air bubbles. Into B add the cell suspension (10-20 ml) in growth medium containing 10% foetal calf serum and allow this to flow into C where it will form a narrow band beneath the medium or PBS already there. Now, without letting air into the system, allow the gradient to flow from A into C. The baffle (D) prevents the solutions squirting into C, thereby disturbing the cell layer. About 500 ml of gradient should be added at 30-40 ml/min. the rate of flow can be controlled by a ‘Rotaflow’ tap Y (Quickfit and Quartz Ltd.). C should contain about 100 ml for every cm of height. Allow to stand for 2-4 h. The lower the temperature, the greater the viscosity and hence the slower the sedimentation. However, if the sedimentation time is long compared to the cell generation time, it is better to stand at 4°C. If too many cells are placed on the gradient ‘streaming’ may occur. Using the three-way tap - Z - allow the separated cells to flow out of C. Discard the medium from the conical part of C and then collect 15 ml fractions.

,Macdonald and Miller (1970) describe an even simpler apparatus and Shall (1973b) describes a mini version where the gradient (15 ml) is formed in a 15 X 1.6 cm test tube, and the cell suspension (3 X lo7 cells) is layered onto this with a wide bore pipette. After standing upright for 50 min at 37°C the topmost 1 ml which contains the smallest and youngest cells may be removed, the sucrose washed off and the cells returned to normal growth medium. This method allows the separation of moderately large cell numbers but sedimentation under unit gravity is a lengthy process and difficulties can be found in keeping the system aseptic. Furthermore, only moderate synchrony is achieved compared with that obtained by selection of mitotic cells.

CH. 11.

CELL SYNCHRONISATION

219

The LACS cell separator is marketed by Medilog (Appendix 3). This simply consists of a separating chamber on which a 360 ml Ficoll gradient (2.5-7.58) is made and a cell suspension is layered onto the gradient through a sieve. The cells are siphoned from the top of the gradient using a conical funnel. The innovation is that the separation occurs while the separating chamber is rotated through 90% (Bont et al., 1979). This allows more cells (2 X 10') to be used and separation to occur in a shorter time. In addition, viscosity changes are minimised by addition of polyethylene oxide which prevents streaming of cells and the gradual slowing on sedimentation of the larger cells as they enter regions of higher density. 11.4.3. Centrifugal elutriation

This involves the use of a special rotor and the adaptation of the centrifuge for constant flow as for the zonal rotor. This is expensive, but once installed, reproducible separations of 108-109cells can be achieved in under an hour and cells of all sizes (rather than just the smallest) are obtained. It is particularly suited to the isolation of G1-phase cells from suspension cultures for which mitotic detachment procedures are not applicable. The separation chamber is kite-shaped with the buffer solution entering at the acute point on the rim of the centrifuge and leaving at the obtuse point towards the centre of rotation (Fig. 11.2). Thus the centrifugal forces on particles within the chamber are countered by the centripetal flow of buffer and particles come to equilibrium within the chamber. When equilibrium has been reached, the samples are pumped out, by increasing the rate of buffer flow, and collected. Meistrich et al. (1977) obtained 3 fractions of L-P59 mouse fibroblasts which were over 90% G1-phase, 70% S-phase cells and 60% G2 M-phase cells, respectively. In fact, almost pure preparations of G1-cells can be obtained (Fig. 11.3) but the later fractions are always contaminated with G1cells such that the best fraction for S-phase contains only 79% S-phase cells together with 19% G1-phase cells. The best G2/M fraction still contains 6% G1-phase cells and at least 25% S-phase

+

220

CELL CULTURE FOR BIOCHEMISTS

Fig. 11.2. Diagram of the elutriator rotor. The cell suspension enters at the acute point of the kite-shaped cell and as the flow rate is increased cells leave (smallest first) towards the centre of the rotor.

cells. Contamination with G1-phase cells may reflect a varied size range for these cells or aggregation of cells. Once the conditions for a particular cell type have been established separations are very reproducible. Initially, with the centrifuge running at, say, 2000 r.p.m. it is important to determine the pump speeds necessary to obtain good fractionation. For example, with MEL cells a pump speed of 22 ml/min will displace G1-phase cells, whereas 36 ml/min is required to displace the largest cells. To some extent this depends on the medium used to displace the cells (usually growth medium from which serum is omitted, partly to reduce cost as 2 1 are required but also because the pump needs to work harder to achieve the necessary flow rates in the presence of serum). It also depends on the temperature. Initially cells should be kept at 37°C so as not to disturb the balance of cells throughout the cycle, and, of course, they must be growing exponentially to achieve high yields of S-phase and G2/Mphase cells. The displacing medium can be at room temperature but the cells should be placed on ice after fractionation until they can be analysed. Each fraction is displaced by 150 ml medium before the pump speed is increased to collect the next fraction. Most of the cells in a fraction are displaced by the first 50 ml but the wash reduces contamination of later fractions. The fractions can be collected aseptically and grown on to yield a population of synchro-

CH. 11.

221

CELL SYNCHRONISATION

Unfractionated

Gl

s

GZM

z

36 Ole

148010 :1 5 " I o

r Fraction 1

G1

2

s

I

89 '10

10 ' I . G,+M = 1 "10

i

G1 1 0 "10 S z 46 O/o G,+M = 44V0

0

50

I00

150

200

FL2

Fig. 11.3. Friend cells fractionated by elutriation and analysed by FACScan. lo8 Friend cells growing in RPMl 5 % FBS were applied at 37OC and 15 ml/min into the elutriator rotor which was spinning at 2000 r.p.m. at 20°C. The pump speed was increased to 22 ml/min to collect the smallest cells, and then stepwise up to 36 ml/min to collect the largest cells. 150 ml were collected for each fraction. The fractions were analysed on a Becton Dickinson FACScan and the figure shows the number of cells plotted against their DNA content for (a) unfractionated cells (b) the smallest cells and (c) the largest cells. Although the smallest-cell fraction is predominantly G1-phase cells, the largest-cell fraction also contains some G1 phase cells which may be a result of cell aggregation or the presence of binucleate cells. The software used the sum of broadened rectangles model to estimate the composition of the fractions. The final positions of G1 and G2+ M cells is indicated by the vertical lines. I would like to thank Drs. Birnie and Conkie of the Beatson Institute for the use of the elutriator and Dr. Campbell and Mr. Alam for help with the FACScan.

+

222

CELL CULTURE FOR BIOCHEMISTS

nously growing cells (and for this the first, G1-phase fraction is most suitable); but the big advantage of centrifugal elutriation is in providing relatively large numbers of cells at different phases of the cell cycle in order to perform biochemical experiments (Adams, 1990). The cells have not been subjected to chemical synchronisation and so artefacts are avoided. Analysis is best carried out by a fluorescence activated cell sorter (see 5 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 8 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 6 12.7.5. The early work on centrifugal elutriation has been reviewed by Pretlow and Pretlow (1979).

11.5. Synchronisation by subculture It is often difficult to avoid introducing some degree of synchronous growth in a culture simply as a results of routine operations. Thus Stubblefield et al. (1967) found that subculture of Don-C cells every 24 h exerted a selection pressure favouring cells with a 12 h generation time and highest mitotic frequencies at about 7 and 19 h. More frequently a cell culture is allowed to leave the exponential growth phase and enter a stationary phase before it is subcultured (see 9 4.2). When this happens the subsequent round of DNA synthesis and cell division is partially synchronised. -

-

Establish cultures of mouse L929 cells and feed every 2 days with Eagle’s medium (Glasgow modificiation) supplemented with 10% calf serum (Fig.4.2). After 10 days, subculture the cells initiating new cultures at 2 X lo5 cells/&. These cultures may be on coverslips for subse-

CELL SYNCHRONISATION

CH. 1 1 .

223

quent autoradiographic analysis of in 75 cm2 flasks for biochemical experimentation. - Label coverslip cultures with tritiated thymidine (5pCi/ml; 5 Ci/mmol) for 30-min periods at various times after subculture. This is conveniently done by adding to 0.5 ml medium lop1 of a solution containing 2.5pCi[ 3H]thymidine at 50pM. - Process the cells for autoradiography (0 12.3) and score the percent of cells labelled and the mitotic index.

400r

h after subculture

Fig. 11.4. DNA synthesis in cells subcultured from stationary phase culture. Mouse L929 cells were subcultured into 5 cm dishes (4x10' cells/5 ml Eagle's MEM containing 10% calf serum). At the indicated times they were incubated for I-h periods with tritiated thymidine and incorporation into DNA measured.

224

CELL CULTURE FOR BIOCHEMISTS

It is found (Fig. 11.4) that few cells enter S-phase before 12h, but by 18 h about 70% of the cells will be making DNA. All the cells divide shortly after 24 h. This is somewhat longer than the normal cycle time and this has been interpreted in two different ways, viz.: 1) Subculturing provides a stimulus and, after a short lag the cells begin to traverse the whole of G1 before passing through S-phase to mitosis. 2) There is a period of about 12 h while cells are growing, and only then is their transition probability increased sufficiently to allow them to pass from the A state to the B-phase and initiate DNA synthesis (see Q 10.4). Although limitations of space are important in causing the cells in crowded cultures to cease growing, limitations in the supply of nutrients are also important (Dulbecco and Elkington, 1973) and form the basis of the next two methods. Allowing cells to enter GO prior to subculture is not appropriate for all cell lines. Thus Swiss mouse embryo fibroblasts (3T3s) are normally maintained in subconfluent culture by splitting them 1 to 3 every 3 days. If they are allowed to reach confluency, they undergo morphological changes (rounding up) and biochemical changes (increased lipid accumulation). That is, they become preadipocytes and develop an increased number of insulin receptors and a sensitivity to ACTH (Serrero et al., 1979).

11.6. Serum deprivation Burk (1970) showed that Syrian hamster cells (BHK21/C13) failed to grow when transferred to medium containing 0.25% serum and could be maintained in a quiescent state for 8 days or more. On readdition of serum no DNA synthesis occurred for 9 h and mitotic peaks were observed at about 23 and 33 h. It appeared the cells had come to rest in G1 and on stimulation showed a lag of about 9 h before entering into exponential growth with a generation time of about 10-12h.

CH. 11.

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225

Adding serum for just 3 h (serum pulse) induces about 50% of the cells to pass through one cycle, and the proportion of committed cells depends almost linearly on the duration of the serum pulse (Brooks, 1976). The recommended procedure for synchronisation of hamster cells is: Suspend BHK21/C13 cells at 106/10 ml Eagle’s Glasgow modification supplemented with 10% tryptose phosphate, 0.05 mM L-serine, 0.1 mM L-ornithine, 0.1 mM hypoxanthine and 0.25% calf serum and plate into 5 cm dishes. With this parent cell line very little DNA synthesis or cell growth occurs, but with the polyoma transformed PYY cells two doublings occur in low serum. - After 100 h add 0.5 ml calf serum per plate to initiate cell growth. -

Temin (1970) and Todaro et al. (1965) showed similar effects for chicken fibroblasts and 3T3 mouse fibroblasts. The low level of serum is important for survival as well as for the subsequent stimulation of DNA synthesis (Cherrington, 1984). A kinetic analysis using time lapse cinematography (Zetterberg and Larsson, 1985) showed that Swiss 3T3 cells were only susceptible to cell cycle arrest in a short period (3-4 h) following mitosis. Even a 1-h exposure to serum-free medium during this time forced the cells into GO from which they required 8 h to return to G1. The length of the postmitotic sensitive phase was very constant at between 3 and 4 h but considerable intercellular variability existed in the duration of the pre S-phase G1 period consistent with a transition probability event (9 10.4). This technique is only appropriate for normal cells which on serum deprivation arrest in G1. Tumour cells often (but not always) arrest in other parts of the cell cycle usually as a result of deficiency in the supply of nutrients rather than as a result of depletion of serum supplied growth factors (Chapter 2). To activate confluent or serum-starved fibroblasts, serum is required to provide competence factors such as PDGF or FGF which make cells competent to synthesise DNA, and progression factors such as EGF and IGFs which allow competent cells to progress

226

CELL CULTURE FOR BIOCHEMISTS

through GO and G1 into S-phase (Scher et al., 1979; Rozengurt, 1980). In fact, there is an ordered series of events required for progression of cells from GO to S-phase, each event being dependent on a different factor. Although cells only require a brief exposure to PDGF for commitment, this can occur in the S-phase of the preceding cycle (Scher et al., 1979). The requirements for epithelial cells are somewhat different (Reiss and Dibble, 1988). Mouse keratinocytes (MK-1 cells) enter a GO-phase within 24 h when confluent cultures are fed a serum-free, low Ca2+ ( < 0.1 mM) medium supplemented with insulin, transferrin and sodium selenate (see § 5.8). Addition of EGF (10 ng/ml) causes cells to enter S-phase after 10-12 h although the percentage of cells responding is not known. Insulin is not essential for this effect but apparently leads to a threefold increase on the rate of DNA synthesis measured 22-24 h after addition of EGF. TGFP(100 pM) completely abolishes the effect of EGF. Seed 5 X lo4 MK-1 cells per cm2 in calcium-free DMEM: Ham’s F12 medium ( 3 : l v/v) supplemented with FBS (5%), insulin (5pg/ml), transferrin (5pg/ml), sodium selenate (5 ng/ml) and EGF (10 ng/ml). - When confluent (3-4 days) replace the medium with medium laclung FBS and EGF. - 24 h later add EGF (10 ng/ml) to induce DNA synthesis 10-12 h later. -

I I . 7. Isoleucine starvation It was observed that CHO cells which had entered a stationary phase in Ham’s F10 medium could be stimulated to undergo a further round of division by changing the medium. The growth limitation was not a serum factor but was traced to a deficiency in isoleucine which is present in Ham’s F10 medium at only about 5-10% its concentration in other media (Ley and Tobey, 1970; Tobey and Ley, 1970).

CH. 11.

CELL SYNCHRONISATION

221

Exponentially growing cells (CHO, L929, BHK21/C13) can be transferred to isoleucine deficient medium containing dialysed serum when they become arrested in G1-phase (Tobey and Ley, 1971; Everhart, 1972; Tobey, 1973; Skoog et al., 1973). The rate of DNA synthesis falls for several hours after transfer to isoleucine deficient medium and the cell number rises by about 30% as those cells in S, G2 and M divide. (Small amounts of isoleucine allow a somewhat larger increase in cell number; Tobey, 1973.) Imbalanced growth does not appear to occur and biosynthetic capacities are maintained at much higher levels than in stationary phase cultures (Enger and Tobey, 1972). Readdition of isoleucine leads to induction of DNA synthesis in most cells starting at 4-5 h and the mitotic index begins to increase at 12 h in CHO cells. Harvest an exponentially growing suspension of CHO cells and wash them in isoleucine free Eagle’s medium. - Plate 7.5 X lO’CHO cells into 5 cm dishes in Eagle’s MEM laclung isoleucine and containing 5% dialysed foetal calf serum. - After overnight incubation replace the medium with fresh medium containing isoleucine (2 X 10-6M) and 10%undialysed foetal calf serum. - If cells are plated onto coverslips [ 3H]thymidine pulses (5pCi/ml) will show the appearance of S-phase cells 4 h after isoleucine addition. -

The mechanism of action of isoleucine starvation is not clear but it is not simply a general deficiency of amino acids as e.g. leucine starvation causes a far more drastic inhibition of cell growth without any synchronising action (Everhart, 1972; Tobey, 1973). Rather it may indicate some subdivision of G1-phase, and the presence of isoleucine appears to be important for the action of IGF (0 11.6) (Scher et al., 1977). This method gives results very comparable to selection of mitotic cells in that almost 100%of the population can be obtained in G1. It is much simpler than mitotic selection and is more readily applied to larger numbers of cells.

228

CELL CULTURE FOR BIOCHEMISTS

11.8. Blockade of S-phase Synchrony at the Gl/S-interphase has been accomplished by interfering with the synthesis of one or more deoxyribonucleoside triphosphate, which are required for DNA synthesis while allowing other cellular procedures such as synthesis of RNA and protein to proceed. If the block is maintained for about 16 h (i.e. T-tS) about 70% of the cells of an exponentially growing culture gather at the beginning of S-phase. In practice it is difficult to know whether the cells actually do make some DNA but at a greatly reduced rate, i.e. whether the cells have entered S-phase or not. Those cells which were caught in S-phase when the blockade was imposed will remain there until the block is reversed when the 70% of the cells at the Gl/S border pass as a synchronised cohort through S-phase and G2 to division. These synchronised cells are often very suitable for studying the events occurring in the S- and G2-phases of the cell cycle. However, they suffer from two disadvantages. 1) Only 70% of the cells are in synchrony - the remaining 30% being up to 6 h ahead of the main group. 2) Events other than those concerned directly with DNA synthesis may proceed in the presence of reagents which block DNA synthesis and certain aspects of the cell's qetabolism may reflect G2 or even G1 activity while DNA synthesis related events are held at the Gl/S-phase boundary. In addition DNA strand breaks occur at hgh frequency in cells blocked in S-phase (Li and Kaminskas, 1984) and some replicons initiate repeatedly (Vassilev and Russev, 1984). This unbalanced growth rapidly leads to cell death if prolonged for more than a generation time (Ruekert and Mueller, 1960). Unbalanced growth will occur in any cells committed to division (0 10.4) yet blocked in one function including those maintained in colcemid for more than a few hours. Moreover, selective blocking of DNA synthesis may have effects which are not apparent until the synchronised cells are released and proceed to the next G1-phase (Firket and Mahieu, 1966; Cress and Gerner, 1977). Schindler et al.

CH. 11.

CELL SYNCHRONISATION

229

(1968) have shown that the duration of G2 is independent of the length of time cells are blocked with amethopterin up to 8 h. The first disadvantage, however, can be overcome by using a double block technique. In this treatment the agent which blocks DNA synthesis is added to cells which have already been treated or selected so that all the cells are in the G1-phase. This ensures collection of all the cells at the Gl/S boundary. The initial selection may be brought about by selecting mitotic cells (see 8 11.2) or by using a population of cells which have just been induced into a growth phase by addition of serum (9 11.6) or a limiting amino acid (0 11.7), by subculture (§ 11.5) or by addition of phytohaemagglutinin (3 6.2.5) for example. The term ‘double block technique’, however, is usually applied to cells where both selection and imposition of the Gl/S-block are by a similar method. Thus high concentrations of thymidine may be used to block DNA synthesis (see § 11.8.3). If a population of cells is treated with 3 mM thymidine for 16 h and then the thymidine removed the 70% of cells arrested at the Gl/S-phase boundary, together with the 30% of cells arrested in S-phase will move around the cell cycle such that 8-10 h later all the cells will be in a sector of G1. If the thymidine block is now reimposed within about 12 h all the cells will have moved to the beginning of S-phase where they will have been arrested. In practice synchronisation at the Gl/S boundary may be achieved by a number of reagents as well as thymidine, e.g. aminopterin, amethopterin, 5-fluorodeoxyuridine or hydroxyurea. 11.8.1. Action of aminopterin and amethopterin (methotrexate)

On addition to cells these inhibitors rapidly bring about inhibition of DNA synthesis. Incorporation of [ 3H]deoxyuridine into DNA is reduced to 3% of control values within 15 min of addition of 10pM aminopterin and even O.lyM reduces incorporation to less than 20% of controls though this takes 45 min (Siegers et al., 1975). Although concentrations of amethopterin between 0.1 and 1pM block synthesis of thymidylate, DNA synthesis continues at a significant rate and pools of thymidylate are slow to dissipate (Adams et al., 1971; Baumunk and Friedman, 1971; Fridland, 1974; Fridland and Brent, 1975).

230

CELL CULTURE FOR BIOCHEMISTS

Folic Acid

A r n i n o p t e r i n ( R : H ) and A r n e l h o p l e r i n ( R z CH,)

Fig. 11.5. Structure of folic acid and its analogues.

Aminopterin and amethopterin are 4-amino analogues of folic acid (Fig. 11.5) and as such are potent inhibitors of the enzyme dihydrofolate reductase (EC 1.5.1.3) (Blakley, 1969). This enzyme catalyses the reduction of folic acid and dihydrofolic acid to tetrahydrofolic acid which is the level of reduction of the active coenzyme involved in many different aspects of single carbon transfer. As is clear from Fig. 11.6, tetrahydrofolate is involved in the metabolism of (a) the amino acids glycine and methionine; (b) the carbon atoms at positions 2 and 8 of the purine ring; (c) the methyl group of thymidine; and (d) indirectly in the synthesis of choline and histidine. In order to concentrate on the effect of the drugs on thymidine metabolism the other actions are bypassed by addition to the culture medium of hypoxanthine or adenosine (30pM or 200pM) and glycine (100pM) in addition to the methionine, histidine and choline normally present. Cells possess considerable pools of tetrahydrofolate, and, except during synthesis of the coenzyme from the vitamin, the only time the enzyme dihydrofolate reductase is required is to regenerate tetrahydrofolate from the dihydrofolate produced during synthesis of dTMP, i.e. during S-phase. Thus only in S-phase cells will the drug have the effect of depleting the pool of tetrahydrofolate and cause the accumulation of dihydrofolate. However, no accumulation of dihydrofolate was found when Chinese hamster cells were grown for 24 h in medium containing 0.25pM amethopterin (McBur-

CH. 1 1

231

CELL SYNCHRONISATION

homocysteine

HCWH

N5 methyl

AT P

f

N’

for my1

(b)

FH,

FH,-

FAICAR

I M P and o t h e r p u r i n e nucleotides

Fig. 11.6. Interconversions of tetrahydrofolate derivatives. FH2 = dihydrofolic acid; FH, = tetrahydrofolic acid; AICAR = 5 aminoimidazole 4-carboxamide ribonucleotide; FAICAR = formyl AICAR; GAR = glycinamide ribonucleotide; FGAR = formyl GAR; Glu = glutamic acid; FIGLU = formimino glutamic acid. (Modified from Mudd and Canton;, 1964.)

ney and Whtmore, 1975) and concentrations of amethopterin up to 2.5pM did not affect glycine synthesis except in cells previously starved of folic acid for 48 h (McBurney and Whitmore, 1975), i.e. in cells lacking a pool of tetrahydrofolate. This led McBurney and Whitmore to suggest a more direct effect of amethopterin in thymidylate and purine synthesis and an action of the drug on thymidylate synthetase has been reported (Borsa and Whitmore, 1969). In Leishmania tropica dihydrofolate reductase and thymidylate synthetase are part of a single enzyme (Meek et al., 1985). McBurney and Whitmore (1975) do show that reduction of folic acid is more sensitive to amethopterin than is inhibition of thymidylate synthesis, and on exposure to low doses (0.02pM) cell growth would become limited by lack of active folate reductase after 48 h. It was

232

CELL CULTURE FOR BIOCHEMISTS

thymidine

4

dUTP UDP-dUDP

t \t

dTMP-wdTDP-dTTPdUMP

t

CDP 4 d C D P d d C M P t - d e o x y c y t i d i n e

I

1

b d

C

T

P

-

1

i

J

DNA

Fig. 11.7. Interconversion of deoxynbonucleotides.

by prolonged exposure to low doses of amethopterin that the mutant lines were selected and this could reconcile the two sets of data. The inhibitory action of the antifolates may be reversed either by changing the medium (Adams, 1969a, b), or more conveniently by additions of thymidine to the medium when dTTP is synthesised using the enzymes thymidine and thymidylate kinase (see Fig.11.7). 11.8.2. Action of 5-fluorodeoxyuridine (5dFUrd)

SdFUrd is an analogue of thymidine and is taken up and phosphorylated in a similar manner to form SdFUMP which is a competitive inhibitor with dUMP of thymidylate synthetase. However, as it may compete with thymidine for uptake and phosphorylation it is not recommended for quantitative studies of DNA synthesis. Its action may be reversed in a similar manner to that of the antifolates. 11.8.3. Action of high concentrations of thymidine Thymidine is taken up by cells and rapidly converted to dTTP, the pool size of which is related to the extracellular thymidine concentration (see § 12.1). At thymidine concentrations as low as 3 X lO-’M this leads to a measurable effect on the rate of DNA synthesis (Cooper et al., 1966). At concentrations above 1 mM inhibition of DNA synthesis is almost complete for some cell lines (Morris and Fischer, 1960; Xeros, 1962; Bootsma et al., 1964;

C H . 11

233

CELL SYNCHRONISATION

log [Thymidine]

M"

Fig. 11.8. Inhibition of cell growth by thymidine. About lo5 cells in 5 ml Eagle's MEM, supplemented with extra vitamins and calf serum, were inoculated into 5 cm dishes and incubated in the presence of the indicated concentrations of thymidine. After 4 days the cells were trypsinised and counted using a Coulter counter. 0, L929; 0,HeLa; A, BSCI; X, X CHO.

Studzinslu and Lambert, 1969). Bostock et al. (1971), however, claim that DNA synthesis proceeds at about one-third the normal rate even in the presence of 2 mM thymidine, and it is obvious from Figs. 11.8 and 11.9 that 5-10 mM thymidine is required to stop cell growth. Inhibition of DNA synthesis is brought about by the action of dTTP as an allosteric inhibitor of ribonucleotide reductase (Reichard et al., 1961; Moore and Hurlbert, 1966; Brown and Reichard, 1969; Kummer et al., 1978). This enzyme is responsible for reducing all four ribonucleoside diphosphates (NDP) to the corresponding deoxyribonucleoside diphosphates (dNDP). It is subject to a complex allosteric control which has been most studied with the bacterial enzyme. Most studies with the mammalian enzyme show it to be similar to the bacterial enzyme (Fig.ll.7). d n P is required as an allosteric activator for the reduction of GDP, and, in turn, dGTP is an allosteric activator for reduction of

234

CELL CULTURE FOR BIOCHEMISTS

Fig. 11.9. Reversal of thymidine inhibition by deoxycytidine. 2000 mouse L929 cells were plated on 200 pl GMEM with NEAA and 10% NBS into the wells of a microtitre plate containing the indicated concentration of thymidine. Some wells (A) also contained aminopterin (4 pM) and hypoxanthine (120 pM) and others ( 0 ) deoxycytidine (20 pM). After 5 days growth in a C02 incubator the cells were fixed and stained with methylene blue as described by Pelletier et al (1988). (a) control cells growing in no, or a low concentration of thymidine, (b) cell growth inhibited by aminopterin, but reversed by 1 pM thymidine. (c) cell growth begins to be inhibited at 0.1 mM thymidine and is strongly inhibited at 2 mM thymidine. (d) the presence of deoxycytidine prevents growth inhibition by 2 mM thymidine. The inhibition seen at 10 mM thymidine is not reversible by deoxycytidine and may result from a drain on the energy reserves of the cells caused by phosphorylation of large amounts of thymidine.

ATP. However, dTTP is an allosteric inhibitor for reduction of CDP and UDP whereas dATP is an allosteric inhibitor for reduction of all four NDPs. It is this complex system which is believed to control the

CH. 11.

CELL SYNCHRONISATION

235

balanced supply of dNTPs for DNA synthesis. As reduction of UDP or CDP is on the pathway to dTTP production control at this point is not unexpected (Fig. 11.7) and DNA synthesis is inhibited by high concentrations of dTTP reducing the supply of dCTP. Apart from removing the inhibitory concentration of thymidine by medium change the block may also be reversed by addition of deoxycytidine at lOpM (Morris and Fischer, 1960; Bjursell and Reichard, 1973) (Fig.ll.9). I I . 8.4. Action of hydroxyurea Hydroxyurea also inhibits DNA synthesis by its action on the M2 subunit of ribonucleotide reductase, but in this case it is the reduction of the purine nucleoside diphosphates which is inhibited and the pool of dTTP rises slightly (Turner et al., 1966; Adams and Lindsay, 1967; Krakoff et al., 1968; Adams et al., 1971; Skoog and Bjursell, 1974; Thelander et al., 1984). What prevents the pool rising dramatically is not clear, but some mechanism comes into play to reduce turnover of the dTTP pool (Nicander and Reichard, 1985). Its action is most satisfactorily reversed by changing the medium for drug free medium.

11.9. Procedure for inducing synchrony at the G1/ S interphase 11.9.I . Isoleucine starvation and hydroxyurea

Plate 7.5 X lo5 washed CHO cells into 5 cm dishes in Eagle’s MEM medium lacking isoleucine and containing 5% dialysed foetal calf serum. - After 8 h incubation change the medium for Eagle’s MEM containing isoleucine, 10% undialysed foetal calf serum and 2 mM hydroxyurea. - After 16 h change the medium again for complete medium lacking hydroxyurea. Most of the cells will be at the Gl/S-interphase and

-

236

CELL CULTURE FOR BIOCHEMISTS

will start to replicate DNA immediately. This may be followed autoradiographically; by total incorporation of [ ’Hlthymidine; and later by the appearance of mitotic cells. 11.9.2. Stationary phase cells and aminopterin -

-

Establish a flask of mouse L929 cells in Eagle’s Glasgow modification containing 10%calf serum. Change the medium on every second day. On day 8 trypsinise the cells and plate 4 X l o 5 cells into 5 cm dishes in the above medium supplemented with aminopterin (2pM) adenosine (200pM) and glycine (100pM). After 16 h reverse the inhibition by addition of thymidine (2 X lO-’M) and deoxycytidine (2 X 10-6M) when the cells will begin to start making DNA, slowly at first but at maximum rate 3 h later.

11.9.3. Double thymidine block

Plate 3 X lo5 HeLa cells in 5 cm dishes in Eagle’s Glasgow modification containing 10% calf serum and 3 mM thymidine. - After 16 h change the medium for fresh medium lacking thymidine. - After a further 8 h add thymidine to a final concentration of 3 mM. - After a further 16 h repeat the second stage when the cells will be released into S-phase synchronously. -

N.B. These times refer to cells with a 24 h generation time. It is not readily applicable to cells with a short generation time in which t S is greater than half T.

I I . 9.4. Comparison of the methods The double thymidine block is the method most frequently referred to in the literature but it is tedious to apply and suffers from the disadvantage that the cells enter S-phase with a pool of dTTP which is decreasing over the first hour or so and this makes estimation of

CH. 11.

CELL SYNCHRONISATION

237

the rate of DNA synthesis difficult (Adams 1969b). Furthermore, it is difficult to use a sufficiently hgh thymidine concentration to block DNA synthesis without producing side effects. The isoleucine/hydroxyurea method also involves two medium changes but the first of these may be replaced by simply adding isoleucine to the deficient medium. The stationary phase and aminopterin method suffers from the disadvantage that it takes a week to prepare the cells but thereafter it is straightforward and DNA synthesis can be followed without interference from endogenous pools (8 12.1.1). Variations and other combinations of these methods are obviously possible, e.g. mitotic selection and thymidine block; aminopterin block reversed with low thymidine followed by a high thymidine block reversed with deoxycytidine. All these methods succeed in accumulating 80-908 of the cells at the Gl/S-interphase and are very suitable for obtaining populations of S-phase cells for study. They are readily scaled up to the level of roller bottles though at this scale the amount of thymidine required for a double block is considerable (0.5 g/roller bottle). Concentrated solutions of thymidine can be made up and sterilised by autoclaving but most of the other solutions should be filter sterilised.

11.10. Synchronisation in G2 The GZphase of the cell cycle is perhaps the most difficult to study as it is the most difficult phase in which to obtain a synchronised cell population. This is because, if cells are synchronised b y selection at mitosis or accumulation at the Gl/S boundary, by the time they reach G2 much of the synchrony has been lost. T h s is because of the dispersion forces arising from the different rates at whch individual cells in a population traverse the cycle. G2 populations are always contaminated with cells in other phases of the cycle and the maximum fractions of Chinese hamster (CHO) cells obtainable in G2 are 0.7 by double thymidine block and 0.4 by mitotic selection (Enger et al., 1968).

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CELL CULTURE FOR BIOCHEMISTS

Although most cultured cell populations come to rest in G1, i.e. between cell division and DNA synthesis, a proportion of mouse ear epidermal cells are thought to be arrested in G2 (Gelfant, 1959, 1963), although recent evidence casts doubt on these conclusions (Sauerborn et al., 1978). There is some evidence that human embryonic fibroblasts maintained in culture for 48 passages (i.e. in the terminal phase) may arrest in G2 (Maciero-Coelho et al., 1966) but t k s e are so abnormal as to be of no value in studies of G2. As mentioned in 6 11.6, some tumour cells arrest in G2 on medium exhaustion, but again many metabolic processes are affected and the cells cannot be considered as typical of G2-phase cells. One way of obtaining a population of G2-cells is to have a mutant with a temperature sensitive stage in G2-phase, and Basilico (1978) has described a procedure which should select for such temperature sensitive mutants.

11.11. Synchronisation in M The selection of mitotic cells has been described in 0 11.2 and the proportion of cells in mitosis can be increased by use of the mitotic blocking agents colcemid or preferably nocodazole. The combination of a single thymidine block for 15-16 h followed by a 4-5 h in 0.04pg/ml nocodazole gives a yield of 25-30% when mitotic cells are harvested (Zieve et al., 1980) and further mitotic cells can be obtained with subsequent shakings.

CHAPTER 12

Use of radioactive isotopes in cell culture Cell cultures offer many advantages over intact animals when it comes to incorporation of radioactive tracers and studying the effects of drugs or hormones. Thus the tracer or drug may be added and removed at known times, its extracellular concentration and specific activity maintained constant and there is no interference in its metabolism by cells of other organs. This does not mean to say, however, that there are no pitfalls to the use of isotopes in cell cultures.

12.1. Estimation of rates of DNA synthesis Estimates of the rate of DNA synthesis in a cell population or in individual cells may be required as a measure of the rate of cell growth, or for cell cycle studies, or to satisfy a basic interest in DNA metabolism. However, on addition of tritiated thymidine to cells in culture a number of problems arise. 1) For incorporation into DNA, the thymidine must be taken up by the cell and phosphorylated stepwise:

Thymidine + dTMP

-j

dTDP + dTTP + DNA

(12.1)

Should the cell have a permeability barrier or should the various kinases be rate-limiting, then the incorporation would not reflect the rate of DNA synthesis. Rather, incorporation would be dependent on the intermediate, rate-limiting enzyme. Uptake is extremely rapid and equilibration of extracellular and intracellular thymidine occurs within 15 sec (Wohlhueter et al, 1979). 239

240

CELL CULTURE FOR BIOCHEMISTS

However, the thymidylate kinases are absent from non-growing cells and thymidine kinase vanes dramatically throughtout the cell cycle (Stubblefield and Dennis, 1976). Thus phosphorylation may well be rate-limiting, except during S- and GZphase, and evidence suggests that there is little phosphorylation outside these phases (Adams 1969a). During S-phase, however, the kinases appear not to be rate-limiting and analysis of the acid soluble pool shows it to be predominantly dITTP (Gentry et al., 1965; Adams 1969a). 2) Within 10 min of addition of tntiated thymidine to a cell culture the amount of 3H in the acid soluble pool (intracellular dTMP, dTDP and predominarnly dTTP) reaches an equilibrium level (Fig. 12.1) (Gentry et al., 1965; Adams, 1969a). Variations in the concentration of thymidine added to the medium affect its uptake, its phosphorylation and the pool size of dTTP. The pool appears to expand indefinitely as the extracellular concentra-

20 -

P--'-"'-'

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16-

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14-

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-,"

12-

E

10-

t

-=

642-

0-

-.o-.-.-, / A S

-B

/' / /

I

/

I

a

8

-.

/

I

/ O

b

/

/

//

/

/

//kDNA

/

0

2yc ' H T (5yM)

/

.I

9 0 '

6

10

4

20

30

40

50

60

Time ( m i n )

Fig. 12.1. Equilibration of tntiated thymidine with the acid-soluble pool and incorporation into DNA. Mouse L929 cells, growing in 5 crn dishes, were incubated with ['Hlthymidine (0.7pCi/mI; 5pM) for the indicated times after which the cells were quickly washed three times with ice-cold BSS. The acid-soluble material (AS) was extracted into cold 5% TCA and after further acid and ethanol washes the cells were solubilised in 0.3 N NaOH and incorporation into DNA measured. (Reproduced from Adams, 1969a. with kind permission of the publisher.)

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

241

i

01

0

I

I

I

I

I

1

2

3

4

5

log,o phymidind x 10' M

Fig. 12.2. Relationship between the concentration of extracellular thymidine and the exogenously derived nuclear pool of dTTP. Mouse L929 cells were incubated for 30 min with [3H]thymidine at the concentrations indicated and the acid-soluble pool extracted (see Fig. 12.1). The concentration of dTTP is calculated on the basis that the radioactive acid soluble pool is completely ('HIdTTP and that it is located in the nuclei of S-phase cells. The volume of a nucleus is taken as 500pm3. (Reproduced from Adams, 1969a with kind permission of the publisher.)

tion of thymidine increases up to 10 mM (Fig. 12.2), which shows that the kinases are present in vast excess (Cleaver and Holford, 1965; Gentry et al., 1965; Cooper et al., 1966; Adams, 1969a; Stimac et al., 1977). Although there is obviously a through-put such that [ 3H]dTTP is being formed and removed by DNA synthesis it appears that the size of the radioactive deoxythymidylate pool is determined by the concentration of extracellular thymidine (Fig. 12.2). This may come about by a combination of forward promotion of thymidine kinase by its substrate and feedback inhibition by d l T P (Ives et al., 1963). The size of the pool remains constant once equilibrium has been reached until extracellular thymidine levels begin to fall. Thls will happen within a few hours when the concentration of extracellular thymidine is 1OP6Mand even at lo-' M 10%may be utilised within

242

CELL CULTURE FOR BIOCHEMISTS

24 h. Thus, if lo6 cells are doubling every 24 h they will require approximately:

lo6 x 9 = 3.3 x lo6 pg thymidine = lo4 pmol

thymidine

(12.2)

This means that if the only source of thymidine is that supplied in the medium 5 ml of 10-6M thymidine which supplies 5 X lo3 pmol is sufficient to support lo6 cells growing exponentially for less than 12 h or t = - Tcu

DN

(12.3)

where t = time in hours until thymidine exhausted; D = DNA content in pg/cell; N = number of cells in millions; u = volume of medium in ml; c = thymidine concentration in nmol/ml (pM); T = generation time (h). For quantitative work it is important to use a high enough concentration of thymidine such that it remains constant throughout the incubation. 3) The pool appears to expand indefinitely as the extracellular concentration of thymidine increases up to 10 mM (Fig. 12.2), whch shows that the kinases are present in vast excess (Cleaver and Holford, 1965;. Gentry et al., 1965; Cooper et al., 1966; Adams, 1969a; Stimac et al., 1977). dTTP is also synthesized endogenously. The pathway involves ribonucleotide reductase. This enzyme is subject to allosteric control and one of the controlling elements is dTTP which at high concentrations inhibits the reduction of CDP and UDP thus leading to a fall in the pool size of dCTP. This, in turn, leads to inhibition of DNA synthesis (Figs. 11.7 and 11.8), an inhibition which may be reversed by addition to the growth medium of deoxycytidine at 5-10pM (Fig. 11.9). This inhibition is detectable when thymidine is added to the grown medium at about 10-6M and becomes absolute above 3 mM, and forms the basis of one method of synchronising cells (see Chapter 11).

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

243

4) The endogenously synthesised d l T P dilutes the specific activity of the [ 3H]dTTP formed from the added [ 3H]thymidine. Thus on adding tritiated thymidine at 3 X 10-*M most of the DNA thymine is synthesised by the endogenous or de novo pathway, but when the [3H]thymidine concentration in the medium is raised to 0.3 mM it contributes 90%or more of the DNA thymine (Cleaver and Holford, 1965; Cooper et al., 1966; Cleaver, 1967). As the specific activity of [3H]dTTP is one of the factors which determine the amount of radioactivity incorporated into DNA (either total counts/min or grain counts) and as this varies (a) with external thymidine concentration and (b) with the state of the cells, the quantitative estimation of rates of DNA synthesis is full of pitfalls. 5) The concentration of endogenously synthesised d7TP varies during the cell cycle. Thus, in resting cells and cells in the G1-phase the pools of d?TP are low (about 3 pmol/106 cells) but these increase throughout S-phase and G2 reaching a maximum at mitosis (Adams et al., 1971; Skoog et al., 1973; Walter et al., 1973). Ths dTTP pool is largely nuclear (Adams, 1969a; Adams et al., 1971; Skoog and Bjursell, 1974) and produces a varying dilution of the specific activity of [ 3H]dTTP arising from exogenous thymidine. There are three ways in which some of these problems may be overcome: 1) Flood the dTTP pool with exogenous [3H]dTTP. 2) Block the endogenous pathway. 3) Make allowance for the contribution of endogenous d l T P to DNA thymine. 12.I,I. Flooding the pool

By measuring the incorporation of thymidine into DNA from [ 3H]thymidine supplied at different concentrations to mouse L cells,

Cleaver (1967) was able to show that, at about 10-5M thymidine, incorporation reached a plateau, and a similar observation has been made for CHO cells (Fig. 12.3). This has been interpreted as showing that at this concentration the contribution of endogenous d l T P to DNA thymine is negligible. Care must be taken, however, that at

244

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I

0.1

0.1

1

0.3

1.0

3.0

1

10

1

30

p h y m i d i n q (pM)

Fig. 12.3. Effect of thymidine concentration on incorporation. CHO cells were incubated with tritiated thymidine supplied at various concentrations and incorporation of radioactivity into DNA measured (for details, see legend to Fig. 12.4).

higher thymidine concentrations inhibition of ribonucleotide reductase is not causing limitation in the supply of dCTP (see Q 11.8.3). Experiments are better carried out at 5 or 10 X 10W5M thymidine in the presence of 5-10pM deoxycytidine. Example

6 X lo5 L929 cells in 5 ml of Eagle’s MEM supplemented with 10% calf serum and buffered with Hepes and incubate overnight. - Without allowing the cells to cool add Sop1 of a solution containing [3H]thymidine (50 Ci/mol) and deoxycytidine so as to give final concentrations of 10-4M thymidine (5pCi/ml) and 10-5M deoxycytidine. To maintain temperature the dishes should be kept in a humidified container inside a 37°C hot room. - After 60 min stop incorporation by washing the cells in the dishes with: (a) cold BSS (twice) (b) cold 5% TCA (4 times) (c) absolute ethanol (twice) - Set up 5 cm dishes containing

CH. 12.

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USE OF RADIOACTIVE ISOTOPES I N CELL CULTURE

245

Air-dry and dissolve the cells in 1.0 m10.3 N NaOH (heat to 37°C if necessary). Rock the dish to mix and remove 0.5 ml to a counting vial. Neutralise with 0.1 ml 1.5 N HCl and add 6 ml of scintillator (e.g. Ecoscint) and count.

12.1.2. Blocking the endogenous pathway

Siegers et al. (1974) showed that in the presence of amethopterin (lOpM), hypoxanthine (30pM) and glycine (100pM) DNA synthesis could be followed by measuring the incorporation of tritiated thymidine present at 3-30pM. The rate of incorporation was linear and calculations based on the specific activity of the tritiated thymidine agreed with the amount of DNA made measured by other methods. Moreover, the presence of amethopterin did not affect the cell proliferation rate, the cell cycle time or the duration of S-phase. The increase with time in the incorporation of radioactivity into DNA is linear between 30 and 90 min of the concomitant addition of drug and tritiated thymidine and during this time the specific activity of the acid soluble nucleotide pool remains constant and identical to that of the supplied [ 3H]thymidine. Example - Set up cultures of hamster CHO cells in glass scintillation vials (2 X l o 5 cells in 1 ml of Eagle’s MEM supplemented wth 10%calf serum, hypoxanthine (30pM) and glycine (lOOpM), and buffered with Hepes buffer. - After overnight incubation add 5Opl of a solution of amethopterin (2 X lO-’M) and tritiated thymidine (2 X lOp4MUI, 0.3mCi/pmol). - At 15 min intervals fix cells in the vials as follows: (a) wash twice with cold BSS (5 ml) (b) wash four times with cold TCA (5 ml) (c) wash twice with ethanol (5 ml) (d) wash with ether (2 ml) and air-dry. - Dissolve the cells in 0.3 ml hyamine hydroxide (heat to 60°C for 10 min if necessary) and add 5 ml toluene scintillator (0.5% diphenyloxazole in toluene) and determine the level of radioactivity.

246

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CELL CULTURE FOR BIOCHEMISTS

Subtract the 30 min radioactivity count from the 90 min count or simply take the 60 min count if the incorporation is linear from zero time.

12.1.3. Allowing for endogenous dTTP (Adams, 1969b)

The problems with flooding the pool are the assumptions generally made that the concentration of thymidine used is (a) sufficient to flood the pool and (b) low enough not to have any deleterious effects on cell growth. The rate of incorporation of radioactivity is dependent on the specific activity of the [ 3H]dTTP which in the two previous methods has been assumed to be the same as that of the supplied [3H]thymidine. As the concentration of [3H] thymidine is increased from low values (i.e. less than 10-6M) the specific activity of the [3H]dTPP pool rises and so does the incorporation of radioactivity into DNA. This is at a time when the true rate of DNA synthesis remains unchanged. Thus the proporation of the d l T P pool which is radioactive, ([ 3H]dTTP(dTTP+ [ 3H]dTTP)), is equal to the proportion of DNA thymine which is radioactive ([ 3H]thymine/total thymine). This equation may be rearranged to give: 1

[3H]thymine

-

[dTTPI 1 total thymine [3Hd-l-l-p]

+

1 total thymine (12.4)

which is the equation for a straight line cutting the ordinate at the reciprocal of the rate of DNA synthesis. Moreover, when the endogenous and exogenous pools of triphosphate (dTTP and [3H]dTTP) are equal, the rate of incorporation of tritium is half maximal, whch enables the concentration of the endogenous dTTP pool to be calculated. The assumption here is that the acid soluble deoxythymidylate pool is predominantly dTTP. This experiment can be done with coverslip cultures or directly in a 24-well TC tray. -

Set up coverslip cultures of mouse L929 cells in a 24- well tissue culture tray. Put 2 X lo5 cells in each well in 0.5 ml Eagle’s

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

247

medium supplemented with 10% calf serum and incubate overnight. - To each well add lop1 of a solution containing 2pCi of [ 3H]thymidine at the following concentrations: 25, 50, 100, 200, 300, 500pM, and 1 mM. - Incubate for 60 min and then remove the coverslips and wash them by dipping them successively into three beakers of ice-cold BSS. - Put the coverslips in a series of scintillation vials, each containing 0.5 ml cold 5 % TCA and stand for 10 min. - Remove the coverslips and wash by dipping successively into 4 beakers of 5% TCA and two of absolute ethanol. - Put the coverslips in a second series of scintillation vials, add 0.3 ml hyamine hydroxide to dissolve the cells and count in 5 ml scintillator to obtain a measure of the radioactivity in DNA ([ 3H]thymine). - To the cold TCA extract from stage 4 above add 5 ml of Ecoscint to obtain a measure of the incorporation of 'H radioactivity into the acid soluble pool ([3H]dTTP). - Convert c.p.m. to d.p.m. and then to pmol taking into account the varying specific activity of the tritiated thymidine. Plot [3H]thymine-1 against [ 3H]dTTP-' and extrapolate to the ordinate to get total thymine-' (Fig. 12.4). The value on the abscissa corresponding to 2 x total thymine is the amount of endogenously synthesised dTTP present in the cells which are making DNA. Wittes and Kidwell (1973) have described a kinetic approach to measuring the pool size of dTTP which involves growing cells continuously in IOpM tritiated thymidine which they found to label about 20% of the DNA thymine residues. They calculated that at this concentration lo6 L929 cells growing in suspension would convert 9.5 pmol extracellular thymidine into dTTP per min during S-phase. 12.1.4, Comparison of the methods

None of the methods is totally satisfactory. The first two involve addition of agents designed to alter the cell metabolism and the third

248

CELL CULTURE FOR BIOCHEMISTS

aJ

c

n

;

c

, .

i

O‘

02

64

66

d8

1’0

1‘2

’H-thymidine in dTTP

1’4

1‘6

1‘8

210

212

( P mol”)

Fig. 12.4. Allowing for endogenous d’ITP. lo5 CHO cells in 0.5 ml Eagle’s MEMGlasgow Modification, supplemented with proline and 10% calf serum, were added to coverslips in each well of a tissue culture tray. About half the cells attached to the coverslips in the wells and 30% of the cells could be shown to be making DNA by autoradiography. lop1 of a solution containing O.SPC~[~HJ thymidine was added to each well to give a final thymidine concentration ranging from 0.1 to 15pM. After 40 min incubation at 37°C the coverslips were removed and processed to estimate incorporation of radioactivity into DNA and acid-soluble material (dTTP). The reciprocals of these values are plotted against each other and the best straight line is drawn through the points. The correlation coefficient is 0.99. The intercept indxates a rate of thymidine incorporation into DNA of 8.9 pmol/llO min/5 X lo4 cells or 0.97pg DNA/h/106 S-phase cells. By measuring the tritiated acid-soluble pool size at half maximal incorporation the size of the endogenous dTTP pool is found to be 62.5 pmol/106 cells. Assuming the volume of lo6 nuclei to be 0 . 1 ~ 1this gives a concentration of 0.63 mM.

is unsuitable for autoradiographic analysis. However, all three methods are attempts to overcome problems which many experimenters prefer to neglect. Any use of tritiated thymidine may cause perturbation of the cell cycle (Hoy et al., 1990). 12.1.5. Application to suspension cultures

When applied to suspension cells the acid washing steps may be done by repeated centrifugation but this is tedious and leads to losses of cellular material unless care is taken. Alternatives involve retaining the cells on glass fibre or cellulose acetate filters in a microanalysis filter holder (Millipore Corp. Ltd.); or the cells may

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

249

be pelleted and then dissolved in a known small volume of 0.3 N NaOH. An aliquot is then allowed to soak into a Whatman 3 MM paper disc (2.5 cm diameter) and many discs may be processed together by washng in a beaker containing 5% TCA (4 times), ethanol (twice) and ether. The cellular material should be dissolved off the paper or glass fibre discs with hyamine hydroxide or NaOH before counting.

12.2. Estimation of rates of RNA and protein synthesis [5-3H)uridineis generally used as a precursor specific for RNA but this is only partially true as significant amounts of radioactivity may enter DNA particularly in the form of cytosine (Adams, 1968; Oldham, 1967). Many of the problems encountered with the use of [3H]thymidine also arise in the use of [3H]uridinebut very little has been done in the search for a rigorous solution. Radioactive amino acids are commonly used to follow protein synthesis. The procedure is in general similar to that used to follow synthesis of DNA, but in this case the cell’s growth medium already contains high concentrations of amino acids and so the added radioactive amino acid acts as a tracer without causing imbalance. However, when it is desired to increase the extent of radioactive incorporation the presence of amino acids in the growth medium may be a disadvantage and their concentration may be reduced. Drastic reduction is obviously deleterious to cell growth (0 11.71 and preliminary experiments must be performed to ascertain what reduction can. be tolerated under the experimental conditions. If a stationary culture of mouse L929 cells is subcultured in the absence of methionine the cells fail to enter S-phase and even the presence of 10% the normal amount of methionine may have serious effects (Turnbull and Adams, 1975). However, once the cells have entered S-phase DNA synthesis will continue in the virtual absence of methionine, but the DNA and probably other polymers made are deficient in methyl groups. Taylor and Stanners (1967) have shown that large polysomes begin to break down within 10 min of reducing the valine content of

250

CELL CULTURE FOR BIOCHEMISTS

medium to 5% normal. This has the result of reducing the rate of protein synthesis measured using a variety of labelled amino acids. Thus although for the same outlay of isotopic amino acid the counts incorporated may rise when depleted medium is used, this may well reflect a reduced incorporation in molar terms.

12.3. Autoradiography Because of the low energy of the /? particles emitted on its decay tritium is ideally suited to high resolution autoradiography. The position of the silver grains is usually 0.1-0.5pm form the source of the /? particles for tritium, whereas it may be up to 290pm for 14 /?particles from C (Cleaver, 1967). The actual range depends on the energy of a particular /? particle and the density of the material through which it must pass, and I4C may be used if resolution is required only down to about 20pm. As well as giving high resolution the low energy of the tritium /? particle poses a problem. As fixed cells are commonly at least 2pm thick, only a proportion of the /? particles will succeed in passing through the cell and reaching the autoradiographic emulsion. Cleaver and Holford (1965) estimated that because of self-absorption of /? particles by the cells and also because at least half the /? particles are emitted in a direction away from the emulsion, it takes 19 disintegrations of tritium to produce one grain in the emulsion. Those /? particles which are emitted at an angle and have to travel through a thicker layer of biological material may never reach the emulsion. One advantage of the low energy of the /? particles from tritium is that those particles reaching the higher density emulsion will be stopped within about 0.15-lpm. Thus as long as the emulsion is thicker than lpm, its actual thickness will have little effect on sensitivity (Doniach and Pelc, 1950). On the other hand, /? particles from I4C will travel, on average, 10pm through emulsion, and so a thicker layer of emulsion while reducing resolution has the effect of increasing the number of grains produced. This has been made use of in double label techniques where DNA has been labelled with tritiated and 14C-labelled

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

251

thymidine and the cells covered with two layers of emulsion (Baserga, 1961; Dawson et al., 1962). The lower layer contains grains produced by both tritium and 14C p particles but only the latter penetrate to form grains in the upper layer. By focussing separately on the two layers only those cells labelled with 14Cmay be detected. This method has been used to measure the passage of cells in and out of S-phase. A booklet is produced by Amersham Int. plc., giving valuable hints on autoradiography. 12.3.1. Fixation and staining

Usually autoradiography is used to detect high molecular weight compounds which are acid or ethanol insoluble, e.g. DNA. These have been made radioactive by incorporation of a low molecular weight, acid soluble precursor and the fixation process must ensure that excess precursor is removed. In order to avoid precipitation of serum protein the cells should be rinsed in cold BSS or PBS (Appendix 1) prior to fixation. Suitable fixatives are Carnoy’s fluid (Appendix 2) or simply 10% methanol, 10% acetic acid. Equally suitable for most purposes is 5% TCA. These latter fixatives will solubilise some proteins (e.g. histones and HMG proteins) and ice cold, 25% TCA may be preferable when it is important these be retained. The acid should be removed by rinsing in ethanol prior to air drying. Staining of cells may be done before or after the autoradiography and Giemsa (Appendix 2) is generally recommended. Some stains, e.g. aceto-orcein will remove silver grains and have to be used before covering the cells with emulsion. 12.3.2. Emulsions

The sensitivity and resolution of an autoradiograph depend on the emulsion used. Emulsions come in two forms: (a) as a gel which needs to be melted and diluted for use by a dipping procedure, and (b) as a preformed sheet which needs to be stripped from a glass plate, floated on a water surface and transferred to the specimen.

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CELL CULTURE FOR BIOCHEMISTS

Examples of the former are Ilford L4, Kodak NTB3 and Amersham’s LM1 and of the latter, Kodak ARlO (Appendix 3). The grain size of these emulsions varies from 0.12pm (Ilford L4) up to about 0.4pm (Kodak AR10) and each p particle hitting the emulsion produces from about 0.5 grains (Kodak films) to 1.3 grains (Ilford L4) (Caro, 1966). Although the use of stripping film was very popular, it is a more difficult and time-consuming procedure and offers no real benefit for tritium autoradiography. However, the even layer of film obtained is an advantage in quantitative autoradiography of I4Clabelled compounds. 12.3.3. Stripping film

1) Coverslips covered with radioactively labelled cells are fixed, cells uppermost, to slides using DePex mounting medium (Gurr, Appendix 3). For best results the slides should be ‘subbed’, i.e. dipped into a 0.5% solution of gelatin containing 0.05% chrom alum and allowed to dry. 2) All further work must be carried out in the dark room using a red safe light. 3) Take a plate of stripping film (12.1 X 16.5 cm) and cut it with a razor blade. Cut 1 cm inside and parallel to each edge and discard the narrow strips. Cut the central portion into eight squares 4) Within a few minutes of cutting the pieces curl up. They are then removed with a scalpel and forceps and turned over onto the surface of clean distilled water at 20°C. 5) Leave the film for 3 min during which time it swells and expands. 6 ) Immerse the slide bearing the cells below the water surface and withdraw it so that the film drapes itself about the slide. The long edges of the film fold round underneath the slide. 7) Drain and dry with a stream of cold air. 8) Expose and develop as indicated below. One major difficulty with stripping film is that the film may move relative to the specimen during processing. This is usually avoided if the swelling (stage 5) and drying processes are adequate.

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

253

12.3.4. Liquid emulsion

Fix the cell bearing coverslips to slides as above. The use of subbed slides is unnecessary. In the dark room thaw the emulsion by standing the bottle in a water bath at 40°C. As this is a lengthy process and one which it is best not to repeat too often on one batch of emulsion, it is preferable to scoop out 5-10 g quantities of the solid gel into universal containers, which should be stored separately. When required one container may be thawed. Dilute the liquid emulsion with one or two volumes of distilled water. Avoid producing bubbles by shaking or vigorous stirring. (LM1 is supplied in diluted form.) Dip the slides into the diluted emulsion. If the coverslip is attached to one end of the slide the depth of liquid emulsion need be no more than 2-3 cm. Drain off excess emulsion and dry in a stream of cold air. Horizontal drying produces a thicker layer of emulsion. If a more dilute emulsion is used then drying in a vertical position may produce a layer of emulsion less than 1pm thick. Autoradiographs are best exposed in a light tight box for 3-7 days. Exposure for longer periods is reported to cause fading of the latent image, unless the autoradiographs are thoroughly dry and preferably stored in an oxygen free atmosphere at low temperature. This may be ahieved by placing a small bag of desiccant in the light tight box along with a lump of solid CO,. The whole may be sealed with tape and stored in a refrigerator. N.B. ensure that no high energy radioactive source, e.g. 32 P, is also in the refrigerator. However, if the layer of emulsion is more than lpm thick the grain counts will increase in a linear fashion up to 9 days even when the slides are stored in air at room temperature (Fig. 12.5). After exposure immerse the slides in Kodak D19 developer or Ilford Penisol at 20°C for 3-5 min. The former may be prepared by dissolving the following in the order given:

2.2 g metol (Ilford Ltd.; Appendix 3)

254

CELL CULTURE FOR BIOCHEMISTS

Autoradiographic e x p o s u r e ( d a y s ) Fig. 12.5. Effect of length of autoradiographic exposure on grain count. BHK21/C13 cells labelled with ['Hluridine were covered with Ilford LA emulsion (diluted with an equal volume of water) dried in a horizontal position and exposed in air at room temperature for the indicated times. (Courtesy of K. Shaw and Dr. J.D. Pitts.)

144 g hydroquinone 48 g anhydrous sodium carbonate 4.0 g potassium bromide

Make up to 1 1 with distilled water and store at 4°C in FULL bottles (to prevent oxidation). Ensure that the developer is warmed to 20°C before use. 8) Fix in Amfix (May and Baker; Appendix 3) diluted with two parts of water for 5 min or twice the clearing time and rinse in tap water. 9) Stain with Giemsa for 3 min and wash thoroughly with tap water. Air-dry. 10) Fix a second clean coverslip onto the specimen using DePex. Unless the labelling is heavy it is usually necessary to use oil immersion optics.

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

255

Although these are the recommended developer and fixer, I have used Kodak LX24 X-ray film developer and Kodak FX-40 X-ray film fixer with equally good results. 12.3.5. Autoradiography in dishes

Cells growing directly in dishes ( 5 cm Petri dishes) may be labelled with a radioactive precursor and fixed in a similar way to cells on coverslips. One ml of diluted liquid emulsion is then added and the material exposed (without the lid) and developed as indicated above. 12.3.6. The value of grain counting

Autoradiographic analysis is the only method which can determine the proportion of cells incorporating a radioactive precursor and the site of that incorporation. Thus, tritiated thymidine is incorporated into DNA in the nuclei of those cells in S-phase and tritiated hypoxanthine appears first in the nucleus and later in the cytoplasm of cells with HPRT but not in mutants lacking the enzyme (see 9 13.2). Grain counting, however, as a quantitative measure of the rate or extent of a given process, such as DNA synthesis, suffers from many of the same disadvantages as does measurement of the total incorporation of radioactivity by a culture (see 9 12.1). Thus [3H]thymidine is diluted by endogenous pools of dTTP which may differ in extent among cells. In addition, the lack of an accurate figure relating grain count to disintegrations means that, at best, grain counts are a relative measure of rates of synthesis of macromolecules. However, grain counting is the only way in which rates of incorporation by individual cells may be compared. Incubation of cells with [ 3H]thymidine not only reveals that only a proportion of cells are making DNA. It also shows that the number of grains per labelled cell is very variable - far more so than would be expected on statistical grounds. Figure 12.6 shows a distribution of grain counts compared with a Poisson distribution with a mean of 30 grains per cell. The wide distribution is caused partly by cells entering or leaving S-phase during the period of

256

CELL CULTURE FOR BIOCHEMISTS

G r a i n count Fig. 12.6. Grain count distribution. L cells labelled for 10 min with 13H]thymidine (2.5pCi/d;0.36 Ci/mmol) and processed for autoradiography using NTB3 emulsion. Those cells (62% of the total) with one or more grains are recorded. A Poisson distribution with a mean of 30 is included for comparison. (Reproduced from Cleaver, 1967, with kind permission of the author.)

labelling and this may be reduced by shortening the pulse time so that very few cells fall into this category. Another cause of the wide distribution of grain counts is the fact that the rate of DNA synthesis is not constant over S-phase (0 10.3) and the population studied will have cells at all stages of DNA synthesis. 12.3.7. Background grains

Apparent in Fig. 12.6 is the group of ‘unlabelled’ cells. Such cells usually average less than one grain per cell and a similar distribution

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

257

of grains is found over regions of the slide where there are no cells. It is common practice to count the grains over such a control area to get a measure of true background. Higher backgrounds are found: (1)if the emulsion used is old or has been stored adjacent to a radioactive source, e.g. 32P; (2) if exposure times are long; or (3) if removal of tritiated precursors or acid soluble components has been inadequate. 12.3.8. Autoradiography of water-soluble cell components

In the previously described methods the radioactive precursor molecules and acid soluble intermediates have been rigorously removed to reduce background grains so that incorporation has been measured only into macromolecules. However, a number of procedures have been developed to visualise the localisation of water soluble compounds within cells. As an initial step these methods rely on the efficient removal of extracellular radioactive material by several washes in saline. It is not clear whether such washes also remove intracellular material which is able to diffuse out of the cells under the washing conditions. Thus little tritiated thymidine remains associated with cells washed three times with ice-cold Earle’s BSS but this could mean either (1) there is no intracellular thymidine or (2) intracellular thymidine rapidly diffuses out of cells washed with BSS. Thus the autoradiographic procedures described below will detect charged molecules which are generally extracted from cells only with acid fixatives, e.g. thymidine phosphates, glucose phosphate, etc. 12.3.8.1. Cell fixation The method used depends largely on whether precise localisation is required or whether a certain amount of diffusion of the soluble compounds can be tolerated or is required. In the latter case rapid air drying (at 37OC) of cells labelled with tritiated thymidine allows thymidine phosphates to diffuse a little way to form a halo around the nucleus (Adams, 1969a). For precise localisation it is necessary to freeze the cells in isopentane or freon held at liquid nitrogen temperature and then to subject them to lyophilisation. This drying

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process is very rapid as the cells are so thin and may be done by placing the slides on a metal block precooled in liquid nitrogen and placing the block in a desiccator attached to a vacuum pump. Alternatively, the slides may be placed in a tube held in a salt/ice mixture at -20°C and the tube connected to a vacuum pump. In both cases traps containing methanol/dry ice must be present to trap the water vapour. Rather than being lyophilised cells may be freeze substituted (Pearse, 1953). After treating cells with isopentane at liquid nitrogen temperature they are flooded in several changes of absolute methanol at the temperature of solid CO, for 2-3 h. 12.3.8.2. Covering with emulsion Once again if a certain amount of diffusion can be tolerated the fixed slides may be dipped into liquid emulsion and quickly dried in a horizontal position (Adams, 1969a). Drying vertically leads to a stream of grains trailing away from cells as the soluble radioactive compounds are washed out by the liquid emulsion. Fitzgerald et al. (1961) cooled fixed slides in a refrigerator so that when they were brought out into the warm dark room a layer of condensation formed on the surface. This is sufficient to allow a square of dry stripping film to be stuck to the slide by means of thumb pressure. Although the film sticks at this stage it usually swells on developing, leading to considerable movememnt relative to the cells. To avoid the problems of unswollen stripping film and to reduce diffusion to a minimum, partly dried liquid emulsion can be added to fixed slides (Miller et al., 1964a; Finbow and Pitts, personal communication).

Place a small amount of diluted liquid emulsion in a 9 cm Petri dish at 44°C and allow it to cool until it begins to set. - Dip a wire loop of about 8 cm diameter into the emulsion and withdraw it covered in emulsion. - Place the loop over the slide lying on a horizontal surface. The emulsion should form a neat circle round the slide. - Dry using a cold fan and expose as for normal autoradiography.

-

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

259

12.4. Labelling with bromodeoxyuridine Rather than using [ 3H]thymidine with the inherent problems associated with radioisotopes, S-phase cells can be detected by labelling with bromodeoxyuridine. This analogue is incoporated into DNA in place of thymidine and can be detected using an antibody to the analogue (Thornton and Wells, 1987). Amersham International plc (Appendix 3) sell a kit for this purpose and, by using a peroxidaselinked immunological detection system, the labelling and detection can be complete in three hours. Incorporation of bromodeoxyuridine is enhanced by incubating in the presence of fluorodeoxyuridine (to block endogenous synthesis of thymidine -§ 11.8.2) and access of the antibody to the fixed cells is ensured by partial denaturation or brief nuclease treatment (Goncharoff et al., 1986). After development of the peroxidase reaction cells can be counterstained, for example, with neutral red.

12.5. DNA repair Incorporation of tritiated thymidine into DNA as a result of repair seldom interferes with studies of DNA synthesis. However, following irradiation, or in the presence of certain drugs which suppress DNA replication, the major reason for incorporation of tritiated thymidine may be to help repair DNA. DNA repair is best studied in a system where the background levels of replication are low. Suitable systems are cultures which have come to rest at high density, or unstimulated lymphocyte preparations where less than 1%of the cells are in S-phase. The low levels of replicative incorporation can be further repressed by 1-2 mM hydroxyurea which selectively inhbits replication (Cleaver, 1969b). This selective effect may simply be a result of the very small pools of deoxyribonucleoside triphosphates required for repair. That incorporation occurring under these conditions is a result of DNA repair can be confirmed by labelling with [3H]bromodeoxyuridine in the presence of lpM fluorodoxyuridine and hydroxyurea. The DNA is then isolated and centrifuged to equilibrium

260

CELL CULTURE FOR BIOCHEMISTS

on a gradient of caesium chloride when the label is found in the light, unsubstituted position (Cleaver, 1969a; Abo-Darub, 1977). 12.4.1. Ultraviolet irradiation

For irradiation cells must be present as a monolayer in a plastic (UV transparent) dish or bottle and must be immersed in a minimum

Incubation t i m e ( h )

Fig. 12.7. Time course of [’Hlthymidine incorporation into DNA of U V irradiated, hydroxyurea-treated lymphocytes. 3 x lo6 lymphocytes from actinic keratosis patients (0) or age-matched normal individuals ( 0 ) were incubated with 13H]thymidine (5pCi/ml, 18.5 Ci/mmol) and hydroxyurea (1.5 mM) at 37°C immediately after irradiation (20 J.m-’). After incubating for different times cells were fixed in Carnoy’s fixative and washed with 5% trichloracetic acid and ethanol before counting. (Reproduced from Abo-Darub et al., 1978, with kind permission of the authors and publisher.)

CH. 12.

USE OF RADIOACTIVE ISOTOPES IN CELL CULTURE

261

volume of PBS-A or some other liquid which does not absorb UV light. They are then exposed to light of 254 nm emitted at a dose of about 0.5 J . m - 2 . s - 1 at a suitable distance (Abo-Darub et al., 1978). The energy emission of the lamp may be measured using a chemical actinometer (Harchand and Parker, 1956) which relies on the fact that irradiation of a solution of potassium ferrioxalate releases ferrous ions in an amount proportional to the dose of ultraviolet radiation. After irradiation growth medium is added to the cultures which are quickly returned to the incubator. 12.4.2. Estimation of repair synthesis

Label cells with [3H]thymidine (5pCi/ml, 20 Ci/mmol) in the presence of 1-2 mM hydroxyurea. After the labelling period harvest the cells in the normal way for measuring incorporation into DNA. Alternatively, cells may be processed for autoradiography when the presence of hydroxyurea is not necessary as S-phase cells are readily recognised by their very high grain count. Unirradiated cells should show less than 0.2 grains per cell whle those irradiated at 5 J . rnp2 should show 15-20 grains per cell after exposure for 8 days (AboDarub et al., 1978). Figure 12.7 shows the time course of repair for irradiated lymphocytes from normal subjects and those with actinic keratosis (a disease, prevalent in certain regions, which produces tumours on areas of skin exposed to sunlight). The rate of DNA repair is slower than normal in the lymphocytes from patients with actinic keratosis.

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CHAPTER 13

Cell mutants and cell hybrids Major advantages that bacteria hold for biochemists are the ready availability of mutants coupled with a short generation time. Compared with whole animals, animal cells in culture have a relatively short generation time and so a major effort has gone into the production and selection of mutants of animal cells and their use in the study of somatic cell genetics.

13.I . Auxotrophic mutants Kao and Puck (1968) have developed a general method for production of auxotrophic mutants. This is based on the observation that DNA containing 5-bromodeoxyuridine (BUdr) is sensitive to visible light produced by fluorescent lamps. This can be used to selectively kill prototrophs growing on restrictive media, i.e. media which restrict the growth of auxotrophic (or other) mutants. The cells that later grow out in supplemented media are auxotrophic mutants. The successive steps involved are given below. Grow cells on enriched media containing the mutagen N-methylN '-nitro-N-nitrosoguanidine (MNNG) at OSpg/ml or ethyl methane sulphonate (EMS) at 200pg/ml for 16 h. - Transfer the cells to mutagen-free enriched medium for several generations to allow the growth of both prototrophs and mutant auxotrophs. - Transfer the cells to growth on minimal restrictive medium for sufficient time for pools of essential metabolites to become depleted. - Add BUdr (1OP6M)to the culture. The BUdr is incorporated into the DNA of the prototrophs which are the only cells growing in minimal restrictive medium. -

263

264

CELL CULTURE FOR BIOCHEMISTS

After 2 cell generations remove the BUdr and expose the cells for 30 min to light from a 40 W ‘Cool White’ fluorescent lamp (General Electric). Prototrophs are killed. - Transfer to complete enriched growth medium when clones of auxotrophs appear after 1-2 weeks.

-

13.2. Selection of mutants Mutants defective in particular enzymes can be selected if two conditions are fulfilled: either the enzyme is not essential or an analogue of the normal substrate leads to lethal incorporation. Cells lacking thymidine kinase ( T I C cells) can be isolated by treating cell cultures with high concentrations (30 pgg/ml) of 5bromodeoxyuridine, which kills cells containing the enzyme thymidine kinase due to incorporation of large amounts of the analogue into the cells’ DNA. 5BUdr

SBdUMP thymidine kinase -+

-+

SBdUDP

-+

SBdUTP + DNA

Mutants lacking, or with much reduced levels of the enzyme thymidine kinase survive as this enyzme is not essential. Thus the cell can make dTMP from dUMP using folic acid as the one carbon donor (Fig. 13.1). TK mutants arise at a very much higher frequency than would be expected for a diploid cell and the evidence suggests that while one of the alleles has been mutagenised, the other allele has been

d U MP ; Se r ,n e 5 10 r a hyd rnethylene r~ o f olat e x t

tetrahydrofolate

dihydrofolate

7-i

NADP

d T M P A t h y m i d i n e

NADPH

Fig. 13.1. Synthesis of dTMP: a = thymidine kinase; b = dihydrofolate reductase.

CH. 13.

CELL MUTANTS A N D CELL HYBRIDS

265

silenced by modification of DNA cytosine (Harris, 1982). In such cells the tk gene is readily reactivated by treatment with the demethylating agent, 5-azacytidine (Harris, 1982; Adams and Burdon, 1985). Functional hemizygosity may also arise in cultured cells as a result of chromosomal aberrations (Siminovitch and Thompson, 1978). In a similar way, mutants lacking hypoxanthine phosphoribosyltransferase (HPRT) can be isolated by growing cells in 8azaguanine at 6-thioguanine (3-30pg/ml) (Evans and Vijayalaxmi, 1981). These analogues are incorporated into the purine nucleotide pool and hence into nucleic acids by the HPRT which normally would be involved in the uptake of hypoxanthine or guanine. Once again, only mutant cells lacking the enzyme survive. However, as the gene is X-linked, cells normally contain only one active copy of the gene which explains the high levels of mutation found in vitro and in vivo where males lacking the enzyme suffer from Lesch-Nyhan syndrome (Adams et al., 1986). HPRT is not essential as IMP is synthesised endogenously from smaller precursors. Two steps in the endogenous pathway utilise folic acid to add single carbon units to the growing purine ring (Fig. 13.2). Aminopterin and amethopterin (methotrexate) are 4-amino analogues of folic acid (Fig. 11.3) and interfere with the production of the active folate coenzyme by blocking the enzyme dihydrofolate reductase (reaction b in Fig. 13.1) (see also Fig. 11.6). By growing cells in the presence of increasing concentrations of aminopterin a number of resistant cells lines have been isolated (Hakala and Ishihara, 1962; Littlefield, 1969). These have been characterised as having either an altered permeability to the drug or an altered folate reductase or an increased rate of synthesis and hence increased amounts of the enzyme (Alt et al., 1976), resulting, at least in part, from a selective amplification of the dihydrofolate reductase gene (Alt et al., 1978; Schimke et al., 1988). The problem is considered in more detail in 6 11.8.1. The importance of the antifolates lies in their role in the HAT selection technique (0 13.5) devised by Szybalslu (1962) (see also Szybalski et al., 1962 and Littlefield, 1964) for the isolation of hybrids between mutant cells defective on the one hand in thymidine kinase and on the other hand

266

CELL CULTURE FOR BIOCHEMISTS

N5N1 0 r n e t h y lene

PRPP +

+

ylutarnine

ATP

"H2 FHZ

slycine OeC'NH-R5'P GAR

FH<

i";

c<7 NHLLHO

I

fl*'\NH-R5'P F GAR

1

Fig. 13.2. Synthesis of IMP. c = Hypoxanthine phosphoribosyl transferase (HPRT); GAR = glycinamide ribonucleotide; FGAR = formyl glycinamide ribonucleotide; PRPP = phosphoribosyl pyrophosphate; AICAR = 5 aminoimidazole-4-carboxamide ribonucleotide.

in HPRT (see below). They are also used to suppress the endogenous pathway when labelling cells with radioactive thymidine or hypoxanthine (§ 12.1.1.2), to synchronise cells by depleting the pools of thymidine (6 11.8.1), and in the clinical field as tools in cancer chemotherapy. Lethal synthesis can also be used to obtain DHFR- cells by incubating in the presence of 6[3H]deoxyuridine.In the presence of DHFR and thymidylate synthetase this is converted into [3H]thymidine and i3H]DNA which results in cell death. 13.2.I, Procedure for isolation of TK - mutants (Littlefield and Basilico, 1966)

Seed 5 X lo5 BHK21/C13 cells into 100 mm dishes in Dulbecco's modification of Eagle's medium supplemented with 10% calf serum, 10% tryptose phosphate and bromodeoxyuridine at 3.3pg/ml. This kills most of the cells and after 10 days only about 20 cells have survived to form colonies. - Harvest the cells and reseed in the same manner but increase the

-

CH. 13.

CELL MUTANTS AND CELL HYBRIDS

261

bromodeoxyuridine concentration to 30pg/ml. This selects for highly resistant mutants. After 10 days a further 20 or so clones will have grown up which are found to have only 1-38 of the thymidine kinase activity of the parent cells and which incorporate ['4C]thymidine into DNA at 5-7% the rate of the parent cells.

13.3. Temperature sensitive mutants The general method of isolating a temperature sensitive mutant involves exposing a mutagenised cell population to an agent lethal to dividing cells at the non-permissive temperature. That temperature is usually 39"C, but 37.5"C has been found more useful in reducing the number of leaky mutants (i.e. mutants which show a high tendency to revert to normal) (Basilico, 1977, 1978; Talavera and Basilico, 1977). Lethal agents include compounds which lead to unbalanced growth by blocking DNA synthesis, e.g. fluorodeoxyuridine or cytosine arabinoside, or agents which are incorporated into DNA where they have deleterious effects, e.g. high levels of tritiated thymidine or bromodeoxyuridine which can lead to breaks in the DNA on subsequent irradiation (Thompson et al., 1970). In general, the mutants isolated are temperature sensistive for DNA synthesis (Sheinin, 1976; Nishimoto et al., 1978), but Basilico (1978) indicates the following procedures for selecting for G1 and G2 ts mutants. 13.3.1. Selection of GI mutants and S mutants

a) mutagenise the cells as described in 0 13.1), b) synchronise the cells in G1 by isoleucine deprivation (0 11.7), c) release at 39°C (see 6 11.7) in the presence of 5pM 5-fluorodeoxyuridine for 2 days, or d) release at 33°C in the presence of 2 mM hydroxyurea and after

268

CELL CULTURE FOR BIOCHEMISTS

10 h change for medium at 39°C minus hydroxyurea but plus fluorodeoxyuridine. Incubate for 12-16 h. Point c will yield G1 mutants as mutants in other phases will grow in an unbalanced fashion and die. Point d yields S-phase mutants for a similar reason. 13.3.2. Selection of G2 mutants and M mutants

a) mutagenise the cells as described in 0 13.1, b) shift to 39°C for a generation time when all ts mutants will be blocked, c) detach mitotic cells (8 11.2) which will include ts mutants blocked in mitosis, d) incubate at 39°C for a generation time in the presence of fluorodeoxyuridine which by causing unbalanced growth leads to the death of wild type cells (0 11.8). e) reduce the temperature to 33°C and select mitotic cells (0 11.2) over the next hour or two; these will be G2 mutants. In all cases the cells must be cloned (8 7.1) and checked to ensure the selection process has worked satisfactorily.

13.4. Replica plating of animal cells One of the tools which has enabled the bacteriologist to screen thousands of mutants is the ability to transfer large numbers of clones from one dish to another while retaining their orientation. In this way a thousand or more clones can be tested simultaneously for several growth characteristics and those of interest picked out from the original dish. With animal cells the problems are that the cells grow firmly attached to the substratum yet at the same time move about so that a single clone very soon grows to cover a wide area. A method described by Esko and Raetz (1978) claims to circumvent these problems.

CH. 13.

CELL MUTANTS AND CELL HYBRIDS

269

Cell monolayers are treated with the mutagenic agent ethane methane sulphonate (400pg/ml: Eastman Ltd.) and allowed to grow for 3 days. - About 1000 cells are plated into a 10 cm dish containing 15 ml growth medium and incubated for 1 day. - Float a disc of sterile Whatman No. 50 paper on the medium and weight it down with glass beads to form a single even layer. - Change the medium every 2-3 days. - After 7-10 days remove the medium; decant the beads and remove the paper disc to whch over 95% of the cells are attached. - Wash the paper disc in a stream of medium (30 ml) to remove any small clumps of cells. - Float the paper disc (cells down) on a fresh dish of medium, weight down and incubate. - Repeat the last three stages every 3 days when each new dish is a replica of the original. -

The cells on the paper disc are viable and may be used in autoradiographic studies. Alternatively, they may be lysed by freeze-thawing and incubated in enzyme assays. An alternative method of achieving the same end is to plate out the mutagenised cells in a 96-well microtitration tray as for cell cloning. When colonies have grown up they can be harvested and distributed to several more trays using manual or automatic cell harvesters and diluters available from Titertek (Flow Labs. Ltd.).

13.5. Somatic cell hybridisation This is a technique pivotal to the genetic analysis of cultured animal cells and to the production of monoclonal antibodies (see Q 13.6) (Ringertz and Savage, 1976; Campbell, 1984). Although such studies are crude relative to the genetic analysis in bacteria, it is now easy to locate genes to particular chromosomes. Traditionally, such studies have been performed by analysis of the offspring of parents showing particular phenotypic characteristics, but the process was taken to the level of biochemical characteristics

270

CELL CULTURE FOR BIOCHEMISTS

following the observations that under particular conditions two cells in vitro will fuse to yield heterokaryotes, i.e. single cells containing two distinct nuclei. A small proportion of these will multiply indefinitely. The first mitotic division after fusion leads to daughter cells with both sets of chromosomes in the same nucleus. Very often at subsequent divisions chromosomes are lost in ones and twos, probably as a result of frequent mitotic abnormalities until a new stable cell line is formed carrying some chromosomes from each parent (Weiss and Green, 1967; Ruddle, 1973; Giles and Ruddle, 1973). In human/mouse and Chinese hamster/mouse hybrids it is always the mouse chromosomes which are retained (Matsuya and Green, 1969). In the study of cell hybrids it is essential to be able (1) to select for heterokaryotes and (2) to recognise them. The simplest complementation analysis invovles the fusion of cells differing in a single gene mutation. Thus if cells requiring glycine for growth (gly-) are fused with cells which require hypoxanthine for growth (hyp-) then, if the mutations are recessive, the heterokaryons will be able to grow in medium lacking both glycine and hypoxanthine. This provides a selection technique for the fusion products. Similarly, if a mouse cell lacking the enzyme thymidine kinase (cannot grow in the presence of aminopterin + thymidine) is fused with a human cell which lacks the enzyme HPRT (cannot grow in the presence of aminopterin hypoxanthine) then only the fusion products will grow in medium containing hypoxanthine (10-4M), aminopterin (4 x 107M) and thymidine (lop5 M) (HAT medium) (Littlefield and Goldstein, 1970). As stated above, such heterokaryons lose human chromosomes preferentially. However, one chromosome they cannot lose while still retaining the ability to grow on HAT medium is the one carrying the gene for thymidine kinase. By talung a number of clones derived from such a fusion event and comparing their human chromosome complement it soon becomes clear which is the essential chromosome and hence the localisation of the gene for the enzyme thymidine kinase could be established (Weiss and Green, 1967). In ths way it has been shown that HPRT is located on the X-chromosome and much work in the 1970’s developed from such observations (Goss and Harris, 1975; Willecke et al., 1976a, b).

+

CH. 13.

CELL MUTANTS A N D CELL HYBRIDS

271

These techniques using somatic cell genetics to do fine mapping have been largely replaced by advances in in situ hybridisation and analysis of restriction fragment length polymorphisms using specific, cloned gene probes (Adams et al., 1986). Human cells are killed by ouabain, but mouse cells are relatively resistant in that 10-3M is a lethal dose. Mouse human hybrids show intermediate sensitivity and hence HAT medium containing 10-7M ouabain can be used to select for hybrids between any human cell and TK- or HPRT- mouse cells. This enables primarily human cells and other non-selected strains to be used in fusion experiments (Mayhew, 1972; Thompson and Baker, 1973). Other selection systems involve the fusion of proline requiring CHO cells and defective mouse cells in HAT medium lacking proline; and the fusion of normal lymphocytes (which do not grow in vitro) with defective mouse cells (which do not grow in HAT medium). It is this latter technique which has allowed the isolation of clones of cells which will synthesise in vitro large amounts of a single antibody (i.e. a monoclonal antibody) (Kohler, 1982).

13.6. Myeloma culture and monoclonal antibody production In vivo, mature differentiated lymphocytes normally synthesise and secrete immunoglobulin (Ig) molecules. Myelomas (tumours) of plasma cells (plasmacytomas) generally synthesise and may secrete one species of immunoglobulin (a myeloma protein) though myelomas derived from early stages of lymphocyte differentiation may synthesise more than one Ig species (e.g. the line SAMM 368). As myelomas arise from previously stimulated lymphocytes, the specificity of the antibody (the Ig) is unknown. A frequently used myeloma line, MOPC 315, has been shown to produce an Ig with affinity for several of the large number of antigens screened. The nature and usefulness of myelomas have been reviewed by Potter (1972, 1975, 1976) and Rabbitts and Milstein (1977). Plasmacytomas may arise spontaneously or be induced, usually in inbred strains of mice (BALB/c or NZB), by injection of mineral oil

212

CELL CULTURE FOR BIOCHEMISTS

or plastic discs. The tumours are normally passaged as solid or ascitic tumours in mice but may be adapted to in vitro culture, where they are generally grown in suspension although under these conditions they tend to lose some of their Ig synthetic activity. The cells grow rapidly and are hyperdiploid. They are grown in RPMl 1640 (Chapter 5 and Appendix 1) supplemented with 10% foetal calf serum and 1%glutamine. They adhere loosely to the surface but may be grown in a stationary culture or in roller bottles at 2-8 X l o w 5cells/ml. They may be dislodged from the substratum by shaking the bottle. Antibodies are powerful tools when it is necessary to characterise the surface proteins (antigens) of a cell and can be used to mark subpopulations of cells; important particularly with regard to specific cell surface phenomena. The main problem is to raise antibodies specific to individual cell surface molecules. If antisera are raised by immunising one species with cells or subfractions of cells from another, a complex response is induced and the antisera require considerable fractionation and purification. In an attempt to overcome this problem Kohler and Milstein (1975, 1976) have reported the results of fusing a mouse myeloma cell line with spleen cells taken from a mouse immunised with sheep erythrocytes. The hybrids were cloned and clones secreting anti-sheep erythrocyte antibodies selected. Hybridoma production is the initial step in the production of monoclonal antibodies which is now a major bioengineering industry. There are many books devoted to this topic (e.g. Campbell, 1984, in this series). 13.6.I . Isolation of spleen cells

kill the immunised animal and sterilise externally by dousing in 70%ethanol; - remove the spleen aseptically to a dish containing serum-free medium; - release lymphocytes by scraping with two 21-G needles and remove the capsule; - disperse by passing through 21-G and 25-G needles; -

CH. 13

-

CELL MUTANTS AND CELL H Y B R I D S

273

pellet at 800g and wash in serum-free medium. A rat spleen will yield 10' cells.

13.6.2. Harvesting of myeloma cells

Myeloma cells (e.g. the rat Y3.Ag.1.2.3 cell line-Galfre et al., 1979) must be growing exponentially when harvested. They should be pelletted at 800 g and washed in serum-free medium. 2-5 x lo7 myeloma cells are required to fuse to 10' spleen cells and fusion techniques are described in Q 13.7.

13.7. Methods of cell fusion Although cells sometimes fuse spontaneously, the frequency of fusion can be dramatically increased by treatment with a number of reagents. Sendai virus used to be a very common fusogen (Watkins, 1971; Stadler and Adelbert, 1972) but is difficult to control. It has been largely replaced by lysolecithin or polyethyleneglycol (PEG) or by electrofusion. 13.7.I . Cell fusion with lysolecithin

Co-cultivate overnight about 2 x lo5 cells of each type in 4 ml medium. - Remove the medium and wash the cell sheet twice with 4 ml of acetate buffer (0.1 M NaCI; 0.05 M NaAc pH5.8). - Add 4 ml of acetate buffer containing 1OOpg lysolecithin/ml. - After 15 min at 37°C remove the lysolecithin and wash the cell sheet twice with complete medium and finally return to the incubator with 4 ml complete medium (Croce et al., 1971; Keay et al., 1972). -

13.7.2. Cell fusion using polyethyleneglycol

This method devised by Pontecorvo (Davidson et al., 1976; Pontecorvo et al., 1977) is the simplest method and the one in most

274

CELL CULTURE FOR BIOCHEMtSTS

common use for hybridoma production. It is very dependent, however, on the actual cells being fused and the source of PEG. -

-

-

-

-

-

Prepare a mixed suspension of the cells to be used (107-10s each of HPRT- myeloma cells (0 13.6.2) and spleen cells from an immunised animal (9 13.6.1)) and sediment. This may be the most important part of the procedure but the efficiency is increased by the subsequent treatments. Remove the medium and resuspend the cells by gentle tapping. Add 1 ml 50% PEG 1500 plus 5% DMSO over a period of 30 sec with gentle agitation which should be continued for a further minute. (PEG for fusion can be obtained in sterile ampoules from Boehinger Corp. Ltd.; Appendix 3.) Slowly (over l-2min) add 5 ml serum-free medium, followed by a further 5 ml serum-free medium.. Sediment the cells at 800 g and wash in serum-free medium. Resuspend in 5 ml medium containing 20% FBS. Dispense loop1 aliquots into the wells of a 24 well TC tray already containing 0.4 ml medium supplemented with HAT (hypoxanthine, 100pM; aminopterin, 0.4pM; thymidine, 16pM; see fj 13.2) Incubate in a CO, incubator at 37°C. The myeloma cells cannot grow in HAT medium and the spleen cells are non-dividing so only fused cells will grow. Feed weekly by removing half the medium and replacing it with fresh medium containing 20% FBS and HAT. Clones will develop within 7-10 days when the level of serum may be reduced to 10%. They should be cloned as described in 6 7.1.2.

13.7.3. Electrofusion

Electroporation involves rendering the cell membrane porous to molecules such as DNA by means of a series of high voltage, electrical pulses. The pores also induce the fusion of cells (Zimmerman, 1982; Glasey, 1988). Initially the cells are aligned in the alternating electric field and pores form on switching to direct current. Adjacent cells fuse as the electric field gradually decreases.

275

CELL MUTANTS AND CELL HYBRIDS

CH. 13.

Fusion is facilitated by the presence of Ca2+ (0.5 mM) and can be performed with very low numbers of cells and herein lies its advantage over fusion with PEG. It is also independent of cell type. It does require a pulse generator which can be carefully controlled and the fusion method of choice remains that using PEG.

13.8. Cell communication Although neither TK- cells nor HPRT- cells will grow separately in HAT medium it is found that mixed populations of these two mutant lines will grow in HAT medium (see Fig. 13.3).

lo6-

-

n

12

24

36 h

40

60

Fig. 13.3. Metabolic cooperation between BHK21/C3 cells. Growth of BHK-HPRTand BHK-TK- cells separately and in mixed (1:l) culture, in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). HPRT = hypoxanthine phosphoribosyl transferase; TK = thymidine kinase. (Reproduced from Pitts. 1971, with kind permission of the author and publisher.)

216

CELL CULTURE FOR BIOCHEMISTS

T h s metabolic cooperation is not a result of the selection of hybrid cells as it occurs almost instantaneously on mixing in the absence of any fusion agent. It is caused by the exchange of low molecular weight compounds through gap junctions. Thus the TKcells obtain thymidylate from their HPRT- neighbours whch get purine nucleotides in exchange. Other low molecular weight compounds which have been shown to pass gap junctions are tetrahydrofolate (Finbow and Pitts, 1979), and dyes of various sizes up to a molecular weight of 1000 (Pitts and Finbow, 1977; Bennettt, 1973) which suggests that most small molecules are exchangeable between neighbouring cells. For cell communication to occur it is essential that the cells are in contact. Using autoradiography the diffusion of nucleotides is readily apparent only between cells in contact, whereas cells equidistant from a neighbour yet not in actual contact do not communicate. Cell communication may interfere with the selection of hybrids unless the cells are plated at sufficiently low densities that contact is made only occasionally. Alternatively, hybrids can be made using a cell line whch does not communicate, and the L929 cell line is one which has lost this ability together with its HPRT- sub-line (A9) (Pitts, 1971). It has been argued that one advantage of using cultured cells is that each individual cell acts independently thereby enormously increasing the statistical validity of experiments. Obviously if cells are communicating this may no longer be true. An illustration of the effect that cell communication may have is that clones of cells have an average of one uprt allele in four which is active (Turker et al., 1989) yet each cell has only two alleles, i.e. the culture as a whole appears to be responding to levels of AMP which equilibrate between all the cells in the culture. 13.8.1. Grain counting and cell communication

Incubation of a population of cells with tritiated uridine or hypoxanthine leads to incorporation of radioactivity into the RNA of all but the mitotic cells. Analysis of the grain counts shows these to fall into a Poisson distribution, the median point of which corresponds

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to the relative rate of RNA synthesis of the culture. Incubation of HPRT- cells with tritiated hypoxanthine leads only to a low background of grains. Mixed populations of cells, some of which lack HPRT, are readily distinguished when plated at low cell density. However, at higher cell densities where cell communication is possible the HPRT positive cells contribute radioactive nucleotides to their mutant neighbours through gap junctions and the distinction between the two cell types becomes less clear (Pitts and Simms, 1977). It may be resolved using a computer programme which not only reveals the presence of two cell types but also give the mean grain count in each type.

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CHAPTER 14

Viruses 14.1. Introduction Cell culture is the predominant and indispensable tool for virus isolation and cultivation; infectivity assays and vaccine production and testing. Although some viruses are more easily isolated in animals and embryonated eggs, the modem era of virology only began when Enders et al. (1949) showed that poliovirus was able to reproduce in various kinds of human embryonic cells in culture whereas in vivo its multiplication is largely restricted to the neurons in the grey column of the spinal cord. In the early years primay cells were used as viral hosts. Although these have limited growth potential they support the replication of a wide variety of viruses. Continuous cell strains such as mouse L929 or human HeLa cells are now commonly used for virus growth in biochemistry laboratories, but for vaccine production untransformed, diploid cell strains are the preferred hosts. When viruses infect cultured cells they produce characteristic morphological changes. The end result of cellular degenerative processes (the cytopathic effect or CPE) may only be obvious after several week’s growth in the presence of the virus but in other cases is obvious after as little as 12 h. The details of the morphological changes vary for different viruses and include complete destruction of the cell monolayer, regions of rounded or fused cells surrounding clear, cell-free areas (plaques) and regions showing haemadsorption (9 14.3.3). If, rather than a productive infection, the virus brings about cellular transformation this also produces characteristic changes in cell morphology and growth characteristics. This is discussed in more detail in 0 14.4. 219

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A detailed introduction to animal viruses may be found in Fenner et al. (1974).

14.1,I . Animal virus classification The classification in Table 14.1 shows 16 groups of animal viruses and is based on (a) the nature of the nucleic acid, (b) the structural symmetry of the virus particle, (c) the presence of an envelope, and (d) the size of the virion. The classification is simplified from that of Fraenkel-Conrat (1974) and resembles that of Wildy (1971), but details of classifications differ as precise taxonomic relationships have not been established. 14.1.2. Precautions to be taken when using virus-infected cells

Viruses produce CPEs on cells and are the agents for many diseases in humans and other animals. In addition, many viruses (e.g. oncorna viruses, herpes type 11, adenovirus and polyoma and SV40) are believed to be agents responsible for tumour formation in animals. Moreover, due to their ability to pass through bacteriological filters it is difficult to exclude viruses from uninfected cell cultures if the viruses are present in suspension in the air of the culture room. For these reasons it is recommended practice to take special precautions when using viruses. The precautions suggested below are of a general nature only and should be used when no particular hazard is expected (i.e. for viruses not listed in the Godber report on dangerous pathogens, 1975). As a general rule most viruses have been assigned to hazard group 2 by the Advisory Committee on Dangerous Pathogens (1984) and several to groups 3 and 4. Where particular hazards are known extra precautions may be required, and the investigator should refer to the above reports which publish codes of practice for use in laboratories holding category A pathogens or viruses in hazard groups 3 and 4, i.e. those so dangerous as to present great risks to the health either of laboratory workers or of the human or animal communities such that material containing live organisms should not be accepted knowingly or held at all in the United Kingdom without authorisa-

TABLE14.1 Animal virus classification Group

Nucleic acid ss ds ds ds

Average MW x 106 2 3- 5 20- 25 54- 92

Virion diam. (nm) 20 45- 55

Parvo Papova Adeno Herpes

DNA DNA DNA DNA

Pox

DNA

ds

160-200

RNA

ss

2

300 X 240 x 100 20- 30

Picorna Toga or encephalo (0rtho)myxo

RNA RNA

ss ss

23

Corona

RNA

ss

3

Paramyxo Rhabdo Arena

RNA RNA RNA

SS

1.5

100-300

ss

6

175x 1 5

Leuko (oncorna or retro) Reo

RNA

ss

10- 12

RNA

ds

10

3

10- 80 100-150

50- 70

80- 120

80- 120

85-120

ss

100-120 70- 90

Shape

Symmetry

Spherical Spherical Spherical Roughly spherical Brick-shaped

Icosahedral Icosahedral Icosahedral Icosahedral Complex

Spherical

Icosahedral

Spherical Roughly spherical Roughly spherical Pleomorphic Bullet shaped Roughly spherical ROUgNY spherical

Cubic Helical

Spherical

Icosahedral

k env.

MVM sv40 Ad2 HSV

+

Vaccinia Polio

+ +

Helical

+

Helical Helical Helical

+ + + +

Complex

e.g.

Sindbis Flu Murine hepatitis NDV

vsv

Lassa RSV Reo

Based on Fraenkel-Conrat (1974), ss and ds refer to whether the nucleic acid is single stranded or double-stranded. + e m . indicates whether or not the virus is enveloped. The abbreviations for the viruses can be found in Appendix 6.

t 4

2

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tion. Viruses which present particular hazard include Newcastle disease virus; foot and mouth disease virus, vesicular stomatitis virus, smallpox virus, rabies virus, herpes type B virus, etc. However, it cannot be assumed that even viruses such as SV40 present no risk to the human population and the various hazards which may arise in biological laboratories are considered in a book published by Cold Spring Harbor Laboratories (Hellman et al., 1973). In addition to good microbiological practice the following recommendations should be adhered to whenever viruses are being used:

- A special room, or suite of rooms, should be kept for the transfer

-

-

-

-

and growth of cells infected with virus. This room should be adequately ventilated under positive pressure and should contain an 'elbow operated' wash hand basin. No live virus should be removed from this area unless enclosed in a tightly closed container. Precautions should be taken to ensure that the outsides of all vessels are free of virus particles. Special protective clothing (lab. coats which fasten at the side or back) should be available for use in the virus suite. After use these should be put in special bags (Sterilin Ltd.; Appendix 3) for autoclaving. Gloves and eye protection are required when working with some hazard group 2 viruses (e.g. Herpes simplex virus). All medium and glassware that has been in contact with virus should be treated with chloros (0 8.1.1) before removal from the virus suite. All plasticware should be sealed in special bags for autoclaving (Sterilin Ltd.; Appendix 3). Equipment for the storage and processing of virus material should be located within the virus suite. An autoclave, preferably double ended, should be available so that laboratory workers are protected from hazardous materials. Aerosol production should be minimised. When aerosols are produced they should be in a Class 1 microbiological safety cabinet exhausted to the outside air.

Once again it should be stressed that these precautions are not adequate when viruses in hazard groups 3 or 4 are being used.

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283

14.2. Virus production In order to study the virus growth curve a one-step growth cycle is performed. A high multiplicity of infection (m.0.i.) is used to ensure every cell is infected - usually 10 plaque forming units (p.f.u.) per cell is adequate. For virus production, however, the infection is prolonged under conditions where secondary infection can occur and a low m.0.i. is recommended especially where there is a tendency for defective virus particles to be produced. In general, the procedure for infection is the same for all virus:cell combinations. Remove the medium from a cell monolayer and wash the monolayer with BSS or PBS (Appendix 1) to remove inhibitors (antibodies) which may be present in the medium. Apply the virus preparation suspended in a small volume of BSS or PBS and allow 30-60 min for adsorption. Replace or supplement the salt solution with fresh medium. 14.2.1. Procedure for production of herpes simplex, pseudorabies or

EMC virus Seed 12 roller bottles each with 2 X lo7 BHK C13 cells in ETC,, (Eagle’s medium containing 10% tryptose phosphate broth plus 10%calf serum) and grow at 37°C for 3 days. - Infect each bottle with 1 p.f.u. virus per 300 cells in 20 ml serum-free Eagle’s medium. - Incubate at 37°C for 1 h to allow the virus to adsorb to the cell. Add 50 ml ETC, and incubate at 37°C for 2 days (or 32OC for 3 days) for herpes simplex virus, 27-36 h for pseudorabies virus or 24 h for EMC. If medium becomes acid add bicarbonate after one day. - Harvest by shaking to dislodge the cell sheet into the medium. (If the cells do not come off the glass some versene may be added but NOT trypsin.) - Centrifuge at 400g for 10 min to pellet cells. Decant supernatant and spin at 20,OOOg (100,OOOg for EMC) for 90 min; discard this -

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supernatant into chloros and resuspend the pellet in Eagle’s MEM supplemented with 10% tryptose phosphate broth and 5 % calf serum (Appendix 1) (2 ml/bottle). Dispense aliquots into bijoux bottles and store at - 70°C (SUPERNATANT VIRUS). - Disrupt the cell pellet by sonication or freeze-thawing 3 times in an ice/alcohol bath. Spin out the debris at 400g for 10 min and dispense aliquots of this preparation at - 70°C (CELL-ASSOCIATED VIRUS). - Both batches of virus should be tested for bacterial contamination (Chapter 9) by inoculating an aliquot of virus into brain-heart infusion broth (leave at 37°C for one week) and into Saboraud fluid medium (32°C for one week) (Appendix 4). Virus may be purified from these preparations by sedimentation through solutions of high density, e.g. saturated KBr or RbCl (Black et al., 1964) when bands corresponding to virions and empty particles are evident. 14.2.2. Procedure for production of S V40 virus

a) Seed 20-30 9-cm dishes with 2 X lo6 BSCl cells in EFC,, (Eagle’s medium containing 10% foetal bovine serum). b) After one or two days growth infect the cells with 0.01 p.f.u./cell (preferably use virus picked from plaques (see 5 14.3.1) as this is known to be non-defective). c) After growth for a week to 10 days (i.e. when most of the cells show a strong CPE) scrape the cells into the growth medium. Stand at 4°C for 1 h and then centrifuge at 15,OOOg for 30 min. d) Resuspend the cells in 20 ml PBS-A and add pancreatic DNase I (40pg/ml) and pancreatic RNase A (12pg/ml). e) Freeze-thaw 3 times or sonicate and then incubate at 37°C for 30 min. f ) Add deoxycholate to 0.15% and stand at room temperature for 90 min. Centrifuge at 16,OOOg for 30 min at 20°C. g) Remove the supernatant into a Spinco SW27 tube and underlay with 10 ml saturated KBr in PBS-A. Centrifuge 23,000 r.p.m. for 3 h at 20°C. Two bands should be visible in the KBr solution: a

CH. 14.

h) i)

j)

k) 1)

VIRUSES

285

lower band containing virions and an upper band of empty particles. Collect the virion band by puncturing the tube and dialyse against PBS-A (2 x 1 h). Add solid CsCl to a density of 1.34 g/ml and centrifuge 3 ml aliquots (overlayered with paraffin oil) in the Spinco SW50.1 rotor for 18 h at 40,000 r.p.m. at 20°C. The virions band with a buoyant density of 1.34 g/ml. Collect fractions, measuring the buoyant density from refractive index, and the absorbance at 260 and 280 nm to detect the virions and empty particles (which will have relatively low absorbance at 260 nm). Pool the virion containing fractions and dialyse against PBS at 4°C for 2 h. Sterilise by filtration through a 0.22pm filter if required for further infection.

Although this procedure is necessary for production of samples of pure virions it is usually unnecessary if a viral preparation is only required for subsequent infections. In such circumstances the cells should be harvested aseptically and processed to step e, omitting the DNase and RNase. The debris from the disrupted cells is pelleted at 15,OOOg for 30 min and the supernatant used as a source of virus. It should be tested for bacterial contamination with brain-heart infusion broth and Saboraud fluid medium (Appendix 4). The virus should be stored at -7OOC at about 10" p.f.u./ml. In 1967 Hirt developed a method of selectively extracting the small DNA of SV40 and polyoma viruses while sedimenting the larger cellular DNA. Remove the medium from infected cells growing in 9cm dishes and wash the infected monolayer twice with PBS-A (Appendix 1). The medium and washes should be added to chloros to kill any virus. - Add 1 ml 0.6% SDS pH 8.0 containing 20 mM EDTA to each dish. Stand at room temperature for 10-20 min. - Add 0.2 ml5M NaCl to each dish. Tilt to mix and scrape the cell -

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CELL CULTURE FOR BIOCHEMISTS

lysate into a sterile plastic centrifuge tube. Leave at 0-4°C overnight. - Centrifuge 17,OOOg for 30 min at 4°C when cellular DNA sediments along with SDS. The supernatant contains 80%of the viral DNA which can be further purified by CsCl centrifugation. 14.2.3. One-step growth curve of SV40

This may be studied in 9-cm dishes or the wells in a tissue culture tray. - Seed with 2 X lo6 (9-cm dish) or 10’ (TC well) cells in 10 ml or 0.5 ml Eagle’s medium containing non-essential amino acids, penicillin-streptomycin and 10% foetal bovine serum (Appendix 1). - After a day or two of growth remove the medium and add SV40 to give 1 p.f.u./cell (2 ml/dish; 0.1 ml/well). - Incubate at 37°C for 60-90 min to allow time for virus adsorption, overlay with Eagle’s medium containing 5% foetal bovine serum (8 ml/dish; 0.4 ml/well). Viral DNA synthesis is maximal on the 2nd day post-infection. it may be detected on the first day and it is associated with - or

24

h

4s

72

post infection

Fig. 14.1. Time course of SV40 infection. Monkey cells in monolayer culture, infected with SV40 at 1-10 p.f.u. per cell. T anti en may be detected by immunofluorescence, viral DNA synthesis by labelling with [ BHlthymidine followed by separation of viral DNA (by Hirt extraction, SDS gradient centrifugation or agarose gel electrophoresis) and mature virions by infectivity using a plaque assay. (Data from Tooze, 1973; Girard et al., 1975; and Basilico and Zouzias, 1976.)

CH. 14.

VIRUSES

287

slightly preceded by - the production of T-antigen. Mature virions are detected after a short lag (Fig. 14.1). 14.2.4. Sendai virus - production and inactivation 14.2.4.1. Production Sendai virus is grown in the allantoic cavity of hens’ eggs. The method is described by Hams and Watkins (1965). -

-

Take infected allantoic fluid at 8000 haemagglutination U/ml (0 14.3.3) and dilute 1 in lo4 with PBS. Inject 0.1 ml into the allantoic cavity of 10-11- day-old fertile hens’ eggs. Incubate at 37°C for 3 days and then overnight at 4°C. Collect the allantoic fluid and centrifuge at 400g for 10 min. Determine the haemagglutination titre (0 14.3.3). Centrifuge 30,OOOg for 30 min and resuspend the pellet in 1/10 the original volume of Hanks’ BSS. Store at -70°C.

14.2.4.2. Inactivation by UV Place 1 ml concentrated virus on a watch glass and expose for 3 min to UV light from a 15W germicidal lamp at incident radiation of 3000 erg-cm-’ s-’. Mix after 1 min and 2 min (see 9 12.5.2).

-

14.2.4.3. Inactivation by P-propiolactone (BPL) (Neff and Enders, 1968) - Prepare a 10% aqueous solution of BPL immediately before use. - Dilute to 1.3% with isotonic saline containing 1.68%NaHCO,. - Add 1 part diluted BPL to 9 parts Sendai virus (haemagglutination titre between 1 :2000 and 1 : 10,000 (0 14.3.3). - Shake in a tightly sealed container at 4°C for 10 min and then keep at 37°C for 2 h shaking every 10 min. - Keep overnight at 4°C to ensure complete hydrolysis of the BPL. - Inactivated virus may be stored in the presence of 0.5% serum albumin at - 65°C for 5 weeks.

288

CELL CULTURE FOR BIOCHEMISTS

14.3. Virus detection The presence of virus may be recognised and in some cases quantitated by a number of tests, e.g. a) production of characteristic effects on cells which may be used in a plaque assay (see below), b) production by infected cells of virus particles which may be identified using the classification in Table 14.1, i.e. nature of nucleic acid, symmetry of particle, c) production by infected cells of viral nucleic acid which may exhibit a characteristic density on buoyant analysis or a characteristic size on agarose gels or sucrose gradients (Kaplan, 1969; Tegtmeyer, 1972), d) production by infected cells or transformed cells of characteristic antigens which may be recognised by staining with fluorescent antibody (9 14.3.2) or which cause changes in the cell membrane leading to haem adsorption (Deibel and Hotchin, 1961) (9 14.3.3), e) the presence of antigens on virus particles may lead to haemagglutination reactions which can be readily quantised (0 14.3.3), f) production by infected cells of characteristic enzymes or enzymes with properties which readily distinguish them from the corresponding host cell enzymes (Keir et al., 1966; Baltimore and Smoler, 1971; Harada et al., 1975). Some of these tests will now be described. As the number of possible viruses and tests is endless, those chosen simply reflect those with which I am familiar. 14.3.1. Plaque assay

The method depends on infecting a small number of cells in a confluent monolayer. The virus produced in the infected cells will move laterally to infect adjacent cells and various techniques are used to prevent further spreading. The degenerative effect on the cells spreads until a visible area of dead cells (a plaque) is apparent. Staining of the cell sheet makes the colourless plaque more easy to see.

VIRUSES

CH. 14.

289

Usually cell cultures are infected with different dilutions of the viral preparation covering a range of lo4. Thus a viral stock will be diluted in 10-fold increments. 14.3. 1. 1, Viral dilution Set up a series of tubes containing 0.9 ml BSS or PBS or Eagle’s medium without serum and to the first tube add 0.1 ml virus stock. Mix the contents and remove 0.1 ml to the second tube and so on. A solution is required which contains 200-400 infectious units per ml and usually the to dilutions are assayed. 14.3.1.2. Suspension assay Herpes and pseudorabies viruses will infect BHK C13 cells in suspension.

a) b) c) d) e)

f)

g)

h) i) j) k)

Prepare a cell suspension containing 3 x lo6 cells/d. Add 4 ml of suspension to each of 5 universals. Add 0.8 ml of the virus dilutions to each universal. Shake gently on the mechanical shaker at 37°C for 20 min. After 20 min add 12 ml Eagle’s 10% tryptose phosphate broth 10%calf serum (for pseudorabies virus) or Eagle’s + 10%tryptose phosphate broth 5% human serum (for herpes virus) to each universal. Mix and plate 4 m1/50 mm Petri dish. Incubate in a CO, incubator at 37°C. After 2 h add 251-1.1of a heparin solution (10 mg/ml) to the pseudorabies infected cultures. After 28 h (pseudorabies) or 48 h (herpes) remove the medium from the dishes (put is straight into chloros). Fix the infected monolayer with 10% formol saline for 10-20 min . Remove formol saline and stain with Giemsa for 10-20 min. Wash off stain gently under tap water and count the plaques using a low power microscope.

+

+

+

I4.3.I , 3. Monolayer assay For herpes and pseudorabies virus this assay is similar to the

290

CELL CULTURE FOR BIOCHEMISTS

Fig. 14.2. Plaques of herpes simplex virus on a BHK/C13 cell layer. A confluent layer of BHK21/C13 cells was infected with a herpes simplex virus type 2 preparation at different dilutions. On the left few plaques are visible but on the right there are many plaques. (With thanks to Dr. J.B. Clements.)

suspension assay except that infection occurs on a confluent monolayer (Fig. 14.2). - Set up 20 dishes (50 mm) with 3 X lo6 BHK C13 cells per dish. Grow at 37°C for 24 h. - Remove the medium from the dishes and inoculate 0.2 ml of each virus dilution onto each of 4 dishes (include a control mock infected with 0.2 ml BSS). - Allow the virus to adsorb at 37°C for 1 h in the CO, incubator. Rock the dishes from time to time to spread the virus over the surface. - After 1 h add 5 ml Eagle’s medium 10% tryptose phosphate broth + 10% calf serum (pseudorabies) or 5% human serum (herpes) and return to the incubator. - After 2 h add heparin to the pseudorabies virus infected cultures. - Continue as for suspension assay, stages h-k.

+

I 4.3.1.4. Agar overlay assay - Set up 2 0 x 5 0 mm dishes with 2-4X106BHK21 C13 (EMC virus) or BSCl (SV40) cells per dish and grow for 24 h at 37°C to obtain confluent monolayers.

CH. 14.

VIRUSES

291

Remove the medium from each dish and inoculate 0.2 ml of each virus dilution onto each of 4 dishes (also inoculate controls with BSS). - Allow the virus to adsorb at 37°C for 1 h in the CO, incubator. Rock the dishes from time to time to spread the virus over the monolayer. - After adsorption add 5 ml agar overlay medium to each dish. This is made up as follows: -

Mix Eagle’s MEM * Noble agar 2.5% Calf serum (for EMC virus) or Foetal calf serum (for SV40) DEAE dextran (0.5%)

75 ml 25 ml (46°C; see below)

5 ml 1 ml

and equilibrate at 46°C and 50°C. The agar sets shortly after pouring onto the plates at 37°C. Incubate at 37°C for 24 h (EMC virus) or 6 days (SV40). Add 2 ml neutral red overlay medium (see below) directly onto the agar and allow to solidify in total darkness. - Return to the incubator for 2-3 h (or even for several days if the plaques are too small for counting). - Plaques may be seen developing and can be counted using a plate microscope or colony counter.

-

To make up 2.5%Noble agar (Difco Labs.) add 25 g agar powder to 500 ml distilled water and make up to 1 1. Place the flask in a container of boiling water until the agar dissolves. Dispense 25 ml amounts into universal containers with metal caps and autoclave at 15 lb pressure for 15 min. Store at room temperature. To make the neutral red overlay medium first dissolve neutral red to 0.4% in distilled water (heat if necessary) and filter through Green’s Filter Paper No. 9044 (Appendix 3). Bottle in 20 ml amounts and sterilise by autoclaving at 15 lb pressure for 15 min. * The Eagle’s MEM should be made up at 1.3 times the normal concentration (Eagle’sx 1.3).

292

CELL CULTURE FOR BIOCHEMISTS

Store at room temperature. For use in the overlay medium add 2.5 ml to 75 ml Eagle’s X 1.3 and 25 ml 2.5% agar. 14.3.2. Fluorescent antibody techniques

In the direct method an antibody raised against a viral antigen is coupled with a fluorochrome and used to stain infected cells. A positive reaction (a yellow-green colour in the fluorescence microscope) indicates the presence in cells of viral antigens. The method may therefore be used to detect cell transformation when the antibody is directed for instance against the SV40 early antigen or the production of mature virus if the antibody is directed against capsid protein. In the indirect method the antibody is not coupled with the fluorochrome. Rather, after antigen and antibody have reacted in the fixed cell preparation, an anti-gamma globulin antibody conjugated to the fluorochrome is added. This method is more sensitive and eliminates the necessity for conjugating each precious antibody with fluorescent dye. Thus one fluorescent anti-rabbit antibody (raised in sheep against rabbit gamma globulins) will pick out and label any antibody raised in rabbits which has reacted with a viral antigen in the infected or transformed cells. An even more indirect method whch does not require the use of a fluorescence microscope is to use enzyme linked antibodies, e.g. the peroxidase anti-peroxidase (PAP) method of Sternberger et al. (1970). Here, after the antigen has been reacted with a rabbit antibody preparation this is conjugated to sheep anti-rabbit IgG which in turn is conjugated to a complex of rabbit anti-horse-radish peroxidase and horseradish peroxidase. This then reacts with diaminobenzidine and hydrogen peroxide when a brown colour indicates the presence of the antigen. If the second antibody is complexed with biotin it will react with streptavidin. The steptavidin may be labelled with a fluorescent dye or an enzyme such as horseradish peroxidase or alkaline phosphatase which can be detected in situ. Kits to perform such assays are available from Amersham or Bio-Rad Laboratories (Appendix 3).

CH. 14.

VIRUSES

293

14.3.2.1. Preparation of antisera Cells or cell extracts when injected into animals cause the production of antibodies. The procedures are detailed by Clausen (1969) in this series of laboratory manuals, and the animals usually used are rabbits, mice, hamsters, etc. Antisera to viral antigens are best raised in a syngeneic host (i.e. a host of the same genotype as that of the cells in which the virus is grown in vitro) if this is available, e.g. injection of SV28 cells (SV40 transformed BHK21/C13 cells) (Wiblin and Macpherson, 1972) into Syrian hamsters leads to tumour production and antibody to T-antigen is present in the serum. 14.3.2.2. Preparation of globulin fraction - Ammonium sulphate fractionation. A crude fractionation of antisera can be carried out to remove most of the albumin. Either slowly add an equal volume of cold, saturated ammonium sulphate solution (adjusted to pH 7 with ammonia) at 4°C and stir for a further 30 min or dialyse overnight at 4°C against 50 volumes of 50% saturated ammonium sulphate.Centrifuge at 1200g for 20 min at 4°C and dissolve the precipitate in PBS-A and dialyse against PBS-A (three changes of 50 volumes) to remove the ammonium sulphate. - QAE Sephadex fractionation Resuspend the ammonium sulphate pellet in 1 ml PBS pH 6.5 and apply to a 20 ml column of QAE Sephadex A-50 (Pharmacia) equilibrated with the same buffer, which is also used to elute the column. Elution of antibodies may be detected by ELISA or by polyacrylamide gel electrophoresis (Campbell, 1984). - Protein A or Protein G affinity chromatography Immunoglobulins show specific affinity for these proteins which can be obtained complexed to Sepharose (Pharmacia). It is a simple matter so apply antiserum diluted in 20 mM phosphate buffer to a small column and subsequently elute the pure IgG using a glycine buffer pH 2.7. 14.3.2.3. Conjugation of antisera with fluoroscein or rhodamine These are the two common fluorochromes and they are covalently linked to proteins by using activated forms (e.g. the isothiocyanates).

294

CELL CULTURE FOR BIOCHEMISTS

Fluoroscein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) are commercially available from, for example, Sigma Chemical Co. (Appendix 3). More uniform reaction is obtained if the isothiocyanates are first absorbed onto Celite. FITC-Celite is available from Sigma Chemical Co. Ltd., but can easily prepared: a) dissolve 25 mg FITC in 25 ml acetone b) add 500 mg Celite and stir for a few minutes at room temperature c) remove the acetone under vacuum and store dry in a desiccator.

Mix 0.5 ml globulin fraction with an equal volume of carbonatebicarbonate buffer, pH 9.3 (4.4 ml 5.3% sodium carbonate plus 100 ml 4.2% sodium bicarbonate). Add 15 mg FITC-Celite and seal to prevent loss of CO,. Mix gently for 30 min at room temperature. - Remove the Celite by centrifugation (800g, 10 min) and separate the conjugated protein from excess dye on a Sephadex-G25 column (1 X 10 cm) eluted with PBS-A. -

Antisera (and conjugated antisera) may be stored at -20°C or kept at 4°C in the presence of merthiolate (1 part in 10,000) which acts as a preservative. 14.3.2.4. Staining techniques Direct method

a) Grow cells on coverslips and infect with virus (say SV40). b) Remove the coverslips from infected and mock infected cultures and, holding them in forceps or in a micro coverslip carrier, dip them into two washes of PBS-A at room temperature and dry them by dipping into two washes of acetone at 0°C. Air-dry (cells up) on filter paper (take care to identify individual coverslips by their position). c) Place coverslips in a 5 cm dish containing wet filter paper. It is often convenient to stand them on small rubber stoppers. d) Flood each coverslip with 2 drops FITC conjugated SV40 T antigen hamster antiserum; place a lid on the dish and leave at room temperature for 45 min.

CH.14.

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295

e) Wash coverslips in three changes of PBS-A and mount by inverting onto a slide using glycerol as mounting medium. Indirect method

Repeat steps a-c of the direct method above. d’) Flood each coverslip with 2 drops unconjugated SV40 T antigen hamster antiserum; place a lid on the dish and leave at room temperature for 30-45 min. e’) Wash coverslips in three changes of PBS-A and drain for 10 min at room temperature; replace in dish. f) Flood with 2 drops FITC conjugated goat anti-hamster antiserum; replace the lid and leave at room temperature for 30 min. g) Wash coverslips in three changes of PBS-A and mount by inverting onto a slide using glycerol as mounting medium. Examine the coverslips using a fluorescence microscope. 14.3.3. Haemadsorption and haemagglutination Many viruses have antigens which adsorb to and cross-link red blood cells. When the antigens are expressed on the surface of infected cells, these cells will adsorb red blood cells (haemadsorption). Alternatively when virions and red cells interact a network is formed and the suspension agglutinates, i.e. the red cells are precipitated. There is some specificity as to which viruses will agglutinate red cells from which animal, but once the combination is known haemagglutination forms a quick assay for virus titre. Blood is collected in a heparinised syringe and the red cells washed three times in 0.85% saline by sedimentation at 200g for 10 min and resuspension. They are finally resuspended in 200 volumes 0.85% saline. The assay is most conveniently carried out in a microtitre plate (Fig. 3.2) and the dispensing may be done by hand or using automatic equipment available from Titertek (Flow Labs. Ltd.; Appendix 3).

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25p1 of 0.85% saline or PBS is dispensed into a series of wells in the microtitre plate and 25p1 of virus suspension is added to the first well and mixed (1 in 2 dilution). 251-11 is then removed from the first well and mixed in the second well (1 in 4 dilution) and so on to give dilutions up to 1 in 2048. 25p1 of red blood cell suspension is now added, mixed, and left, either at 4"C, room temperature or 37°C until haemagglutination has occurred (1-2 h). In wells where positive agglutination has occurred the red cells have an irregular outline while in negatives and in the control, cells settle in a compact dot in the bottom of the well. The dilution in the last well showing a positive reaction is considered the titre and this dilution has 1 haemagglutinating (HA) unit. The next dilution has 2 HA units and so on.

14.4. Production and testing viral vaccines New cell culture techniques developed in the 1940s led to the isolation of many viruses in virulent and attenuated forms. This helped directly in the production in the 1960s of vaccines for poliomyelitis, measles, mumps and rubella. The major concerns with vaccine production are firstly, is the virus harmless and secondly is the vaccine free of adventitious agents. If the vaccine is produced from a virulent virus it is essential that the virus is completely inactivated before vaccine distribution. If attenuated strains are used the stability of the attenuation must be monitored. In both cases cell cultures are used in tests. Extraneous contamination can be a problem and the use of human serum is discouraged since early batches of yellow fever vaccine became contaminated with hepatitis B virus. For similar reasons normal human diploid fibroblast strains are the preferred cells as they can be shown to be free, not only of contaminating virulent viruses but also of transforming viruses (Furesz et al., 1988). Such cells have replaced primary cells but they must be regularly tested for a) the presence of extraneous agents b) tumorigenicity by both in vivo and in vitro tests c) chromosomal characteristics.

CH. 14.

VIRUSES

291

Following growth of the virus in batch cell culture the culture fluid containing virus particles and cell debris is clarified by lowspeed centrifugation or filtration to remove the latter. The virus preparation (inactivated when required) must then be tested for a) the presence of extraneous agents b) microbial sterility c) virus identity and potency. To circumvent some of these problems antigens produced by recombinant DNA technology often provide the preferred vaccine. Cells are transfected (0 7.5) with a plasmid carrying the gene for a viral surface antigen linked to a strong promoter. The use of single viral antigens as vaccines rules out contamination by unattenuated viruses or by virus which has survived the inactivation process. Mammalian cells are the ideal hosts for the production of such antigens as the viral proteins are folded and modified normally. Furthermore, the antigen gene can be modified to delete regions which would otherwise cause the antigen to be bound to the cell membrane. This allows the secretion of the correctly folded and modified antigen by the transfected cells (Berman et al., 1988). Two examples of such vaccines are the production of a truncated form of one of the HIV surface glycoproteins (gp 130) and a truncated form of HSVI glycoprotein D both of wluch can be isolated from the culture supernatant (Lasky et al., 1984).

14.5. Viral transformation of cells When a virus penetrates a cell instead of causing a productive infection it may transform that cell. For this to happen its genome (or a part of its genome) must integrate into the cellular chromosomes. The site of integration does not appear to be unique and often several copies of the viral DNA are integrated. The following characteristics have been noted in cells transformed by SV40 or polyoma virus (Tooze, 1973). Growth: lugh or indefinite saturation density, reduced serum requirement, growth in agar or Methocel (see Appendix 6), suspension tumour formation on injection into susceptible

298

CELL CULTURE FOR BIOCHEMISTS

animal, not susceptible to contact inhibition of movement, growth in disoriented manner, growth on monolayers of normal cells; Surface: increased agglutinability by plant lectins, changes in glycoprotein and glycolipid composition, foetal antigens revealed, tight junctions missing transplantation, antigen present Evidence of virus: virus specific antigens present viral DNA sequences detected viral mRNA present virus can sometimes be rescued. 14.5.1. Methods of transformation

There are at least four methods of transforming cells with viruses. A) The simplest is that of Macpherson and Montagnier (1964). The cells are infected with the virus in suspension and then plated in 0.33% agar or 1.2% Methocel (Appendix 6) when transformed cells form large colonies and untransformed cells do not grow. Prepare a basal layer of 7 ml of medium (Eagle’s Glasgow modification containing tryptose and calf serum) containing 0.5% Difco Bacto agar in a 6 cm dish. Infect a cell suspension for 1 h at 37°C and add 103-5X lo5 cells in 1.5 ml medium containing 0.3% agar or 1.2% carboxymethyl cellulose (Stoker, 1968) to the preset basal layer. Incubate 7-10 days at 37°C in a CO, incubator when transformed cells alone form colonies. B) Expose subconfluent monolayers of cells to the virus and grow for 2-3 weeks when dense clones of transformed cells grow out of the untransformed monolayer. C) Infect cells and plate out at a low density in low serum medium. Many cells may divide once or twice (abortive infection) but the stably transformed cells will form colonies in the absence of serum factors (Smith et al., 1970). D) The above methods rely on the selection of the transformed cells by making use of one of the properties listed above. Stoker and

CH. 14.

VIRUSES

299

Macpherson (1961) exposed confluent monolayers or cell suspension to virus and then plated the cells at low density. They then picked out transformed colonies by appearance in the absence of any selective pressure (see Fig. 2.1). The number of transformed colonies is directly related to virus dose. When primary hamster kidney cells were exposed to polyoma virus at 96 p.f.u./cell between 1 in 100 and 1 in 5000 cells was transformed.

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CHAPTER 15

Differentiation in cell cultures The study of differentiation in multicellular animals is beset by problems arising from the complex and poorly understood interactions of the various tissues. These interactions can be limited or eliminated in cell culture and a number of systems are described below which are leading to an understanding of the mechanisms of cell and tissue differentiation. This chapter supplements material presented earlier, especially in Chapter 6 .

15.1. Erythroid differentiation of Friend cells The process of erythropoiesis has been reviewed by Harrison (1976, 1977) and Orlun (1978) and more recently Metcalf (1989) has reviewed haemopoiesis from the molecular point of view. Erythroid cells together with the other blood cells are derived from a common haematopoietic stem cell. After commitment to the erythroid lineage the stem cells proliferate for a few generations when they become sensitive to the hormone erythropoietin which increases the proliferation of committed erythroid stem cells and proerythroblasts which then differentiate into mature erythroid cells containing haemoglobin. Studies with normal erythropoietic systems are hampered by the difficulties of obtaining sufficient erythroid cells of specific developmental stages. However, cell strains are available of both human and murine origin which allow the study of the final stages of erythroid development in vitro. Friend cells are murine, virus-transformed, erythroleukaemic (MEL) cells which grow in culture in suspension and exhibit a limited degree of differentiation along the erythroid line (Friend et al., 1966; Patuleia and Friend, 1967). The target cell for the Friend 301

302

CELL CULTURE FOR BIOCHEMISTS

virus is probably a late committed erythroid stem cell. Under normal growth conditions only 1-2% of the cells stain with benzidine (haemoglobin-positive). They appear to be arrested at the proerythroblast stage and are not responsive to erythropoietin. However, on treatment with dimethylsulphoxide (2%), after a 3-day lag the proportion of benzidine-positive cells rises to 96% on the 5th day (Friend et al., 1971), i.e. they undergo a series of changes characteristic of normal erythroid cell differentiation. Marks and Rifkind (1978) have reviewed the characteristics of the induced differentiation and listed the large variety of inducers (e.g. butyric acid, hemin, ouabain) which may be used in place of dimethylsulphoxide. However, not all these inducers produce the same effects in all the clones tested and some DMSO resistant variants exhibit different phenotypes (Harrison et al., 1978). The most commonly used inducers are DMSO and HMBA (hexamethylene bisacetamide). The action of DMSO leads to cessation of cell division and production of globin messenger RNAs (Harrison, 1976; Minty et al., 1978) which is blocked specifically by hydrocortisone (Scher et al., 1978) and is dependent on some event which occurs in the G1-phase in synchronised cells (Gampari et al., 1978) though whether or not DNA synthesis is essential appears open to question (Harrison, 1976). Razin et al. (1986) have shown that within 12-20 h of treatment of the cells with inducer there is a marked fall in the proportion of DNA cytosines methylated but that the level returns to normal prior to globin production. The uninduced cell aleady shows a reorganisation of chromatin around the globin genes typical of globin producing cells (Cohen and Sheffery, 1985; Benezra et al., 1986). In the absence of inducer, Friend cells divide normally and frequently but in the presence of inducer, although a tenfold increase in cell number may occur cell viability is markedly reduced and a terminally differentiated, non-dividing cell population is produced. 15.I . 1. Induction of globin synthesis in Friend cells

MEL cells grow very rapidly and require subculture, by dilution (1 to 10) at least every other day to maintain viability. They can be

CH.15.

DIFFERENTIATION IN CELL CULTURES

303

grown in Eagle's BME supplemented with 15% FBS or, preferably, in RPMI 1640 containing 5% FBS and non-essential amino acids (Appendix 1). Large numbers of G1-phase cells can be obtained using the elutriator (see 6 11.4.3). Place lo6 cells in a 6 cm Petri dish containing 10 ml medium, or lo7 cells in 50 ml medium in a 125 cm2 flask. Add reagent grade DMSO (unsterilised or sterilised by autoclaving in small volumes) to 2% or HMBA to 5 mM. After 5-6 days the cells may be washed in PBS and then lysed in distilled water. By reading the optical density of the clarified supernatant at 415 nm the amount of haemoglobin present ma be estimated. Alternately the cells may be grown in the present of K Fe and radioactive haem extracted and counted to give a measure of haemoglobin synthesis (Freshney, 1975).

15.2. Skin and keratinocytes The differentiation of the epidermal cells in a basal layer of dividing cells and an upper layer or layers of cells which become keratinised and eventually sloughed off appears a relatively simple system and it is made even simpler by the ability to grow keratinocytes in culture. Such an in vitro system can be used not only to study the differentiation of skin and diseases which affect the slun but may also be used to test drugs which affect human epidermis and possibly to produce large amounts of epidermal cells which could be used in skin grafting. Epidermal cells depend for their maintenance and growth on the presence of fibroblasts or their products and when the two cells are present in optimal proportions growth and differentiation proceed very well in culture. However, their lifetime is restricted to 20-50 generations (Hayflick and Moorhead, 1961), which is very much shorter than the in vivo lifetime of a cell of the basal layer of the human epidermis which undergoes 30 divisions per year. The culture lifetime can be increased to about 150 generations by addition of epidermal growth factor (Rheinwald and Green 1977; see 6 2.5) and CAMP levels may also play an important role in growth regulation (Green, 1978).

304

CELL CULTURE FOR BIOCHEMISTS

Described below is a method for growing keratinocytes from skin biopsies using a feeder layer (Rheinwald and Green, 1975). The feeder cells may be 3T3 and BHK21 cells treated with gamma rays or with mitomycin C as described in 8 7.1.4. -

-

-

-

-

-

Dissect the skin biopsies (see 8 6.3) into Dulbecco’s MEM with 10%calf serum (Appendix 1) and mince finely. Stir, in about 10 ml 0.25% trypsin in PBS-A at 37°C. Every 30 min allow lumps of tissue to settle for 1 min and withdraw the supernatant, replacing it with fresh trypsin. Sediment the cells from the supernatant and resuspend in growth medium containing 20% foetal calf serum and hydrocortisone (0.4pg/ml) (this accelerates growth and makes colony morphology more orderly). Mix with a suspension of feeder cells and distribute the cells into dishes. The proportions of the two cell types affect the character of the subsequent growth. The dishes should be left undisturbed as epidermal cells take 2-3 days to attach firmly. Thereafter the medium should be replaced twice weekly. The growth of fibroblasts is largely suppressed but the epidermal cells form colonies of keratinocytes which push back the feeder layer at the periphery. To subculture the keratinocytes first treat the culture with 0.02% EDTA for 15 sec and pipette vigorously to selectively remove the fibroblasts. Then disaggregate the keratinocyte colonies with 0.02% EDTA: 0.05% trypsin (1:1). The keratinocytes may be replated with a fresh feeder layer if required.

If the cultures are not subcultured when they become confluent then cells are shed from an upper layer while the basal layer continues to divided (Green, 1977). Thus the situation closely resembles the stratum corneum in vivo where the basal cells multiply and the non-basal cells undergo differentiation.

CH. 15.

DIFFERENTIATION IN CELL CULTURES

305

15.3. Teratocarcinoma cells Teratomas and teratocarcinomas have been reviewed by Hogan (1977) and Illmensee and Sevens (1979) and were the subject of the 1983 Cold Spring Harbor Conference. Teratomas are tumours arising in the ovary or testis or in early embryos transplanted into extrauterine sites. They consist of a mixture of tissues among which are recognisable skin, nerve, muscle, cartilage, etc. If, interspersed among the recognised tissues, there are undifferentiated embryonal cells, the tumour is called a teratocarcinoma and is recognised by a fast growth rate and by its ability to be transplanted into syngeneic animals (i.e. animals of the same genotype). The stem cells of the teratocarcinoma are embryonal carcinoma (EC) cells which are primitive, pluripotential cells, but which are capable of differentiating into benign somatic tissues. Thus, incorporation of mouse teratocarcinoma cells into the cell mass of early mouse embryos can give rise to completely normal chimeric adults in w h c h the teratocarcinoma cells have been incorporated into many different tissues (Mintz and Illmensee, 1975; Papioannou et al., 1975). It is clear that, in vivo, these cells are able to participate in normal morphogenesis and differentiation, and a considerable amount of work has recently gone into mimicking these events in vitro. A number of cell lines have been established which can be triggered to differentiate rapidly and in a predictable manner following stimulation. Two methods have been developed to establish mouse teratocarcinomas cells in culture. 1) Tumour cells are allowed to attach to gelatin-coated tissue culture dishes (see 6 2.4.1) when many different kinds of cells including embryonal cells grow out. These latter cells may eventually outgrow the differentiated cells and they may then be cloned (Chapter 7) (Rosenthal et al., 1970; Bernstine et al., 1973). Lines isolated in this way tend after some time to lose their ability to differentiate. 2) Tumour cells (ascites or dissociated solid tumours) are plated out on a monolayer of feeder fibroblasts (see Q 7.1.4). After several days many different sorts of differentiated cells may be seen and also nests of embryonal cells. On repeated subculture these cells

306

CELL CULTURE FOR BIOCHEMISTS

will predominate, but it is perhaps easier to isolate them manually and reseed on a feeder layer (Martin and Evans, 1975a). Once isolated the undifferentiated cells ‘remain homogeneous as long as they are subcultured using 0.25% trypsin, 0.05 mM EDTA in PBS (Chapter 4) into Dulbecco’s modified Eagle’s medium (Appendix 1) supplemented with 5-20% bovine serum before they become confluent. If the teratocarcinoma cells become confluent they will begin to differentiate into many different cells types, but this process is not so dramatic as the differentiation which occurs if cells isolated by method 2 are transferred to a vessel without a feeder layer. Then the cells attach very poorly and form clumps in suspension. These clumps remain healthy and quickly differentiate (Evans, 1972) to form an outer layer of endoderm cells. The presence of endoderm can be shown by assaying for the serine protease plasminogen activator which is a marker typical of endoderm cells (Strickland et al., 1976). These aggregates are known as embryoid bodies and after 2-3 days in culture they develop a fluid filled cyst on one side and other signs of differentiation may be seen. If the aggregates are allowed to reattach to tissue culture dishes several lunds of differentiated cells grow out including beating muscle, nerve and glandular tissue (Martin and Evans, 1975b). Bronson et al. (1983) have been able to obtain undifferentiated growth of human EC cells in the absence of feeder layers by seeding at very high cell densities (lo7 cells/25 cm2 flask). Initially subculture of these cells at lower density led to layers of cells of varied mosphology but, after several passages at lower density, the differentiation potential of these cells was lost. Some teratoma cell lines isolated by method 1, e.g. the F9 cell line, have almost lost their capacity to differentiate. However, Strickland and Mahdavi (1978) have shown that differentation may be induced with retinoic acid at lOP9M. Again endoderm formation is detected by formation of plasminogen activator, a- foetoprotein, or by use of immunological markers (Adamson and Grover, 1983). F9 and similar EC cells are sensitive to trypsinisation and this sensitivity can be reduced by treating with a 0.025% trypsin : versene

CH. 15.

DIFFERENTIATION IN CELL CULTURES

307

solution containing protein - usually in the form of chick serum (1%)which does not contain a trypsin inhibitor. Following release from the substrate they should be diluted in growth medium (a 1 : 1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DMEM) (Appendix 2) supplemented with 10% heat inactivated FBS and 1% HEPES and plated out on gelatin coated dishes (0 2.4.1) at less than 105cell/ml When plated onto untreated dished F9 cells proliferate rapidly and form clumps of necrotic spheres by 4-8 days. In the presence of 5 X lO-'M retinoic acid (supplied from a stock of 1 mg/ml in DMSO) proliferation and aggregation occurs but, by about 5 days, embryoid bodies are formed which remain stable for more than 25 days (Adamson and Grover, 1983). It is in the early stages that differentiated functions are first expressed and alpha foetoprotein can be detected in the outer cells of the embryoid bodies at 6-8 days depending on the initial seeding density. There is increased production of laminin at 2 days.

15.4. Differentiation of muscle cells The transition from dividing myoblasts to multinucleate muscle fibres is one of the most striking examples of terminal differentiation which can occur in vitro. The biochemical changes result in the development of an excitable membrane, assembly of the contractile apparatus and the appearance of appropriate enzymes, and have been summarised by Buckingham (1977). The added attraction of the transition from myoblast to myotube is the synchrony with which differentiation occurs in vitro. Myogenesis will occur in primary cultures of skeletal muscle ( e g 0 6.12) but can also be induced in diploid myoblast lines (Richter and Yaffe, 1970) which has allowed the selection of mutants (Chapter 13) that exhibit drug resistance or temperature-sensitive differentiation (Loomis et al., 1973; Somers et al., 1975). Holtzer et al. (1975) and Fiszman and Fuchs (1975) have developed a myoblast line transformed with a temperature-sensitive virus. At the permissive temper-

308

CELL CULTURE FOR BIOCHEMISTS

ature the cells divide continuously but when the temperature is raised differentiation is induced. Myoblasts may differentiate and fuse without undergoing DNA synthesis (Nadel-Ginard, 1978), but fusion does not occur in calcium-free medium. Thus, if chick embryo myoblasts are set up in calcium-free ( < 20pM Ca2+) DMEM containing 5% heat inactivated FBS and 2%chick embryo extract (Appendix 1) the fusion wluch normally starts at around 24 h fails to occur. Addition of 1.4 mM CaCl, after 50 h will now produce synchronised fusion of cells (Wakelam and Pette, 1982).

15.5. Differentiation of adipose cells From the 3T3 cell line originally isolated from mouse embryos (Todaro and Green, 1963) Green and Meuth (1974) have isolated two clones which, on entering the resting state, accumulate large amounts of triglycerides in multiple cytoplasmic droplets. The resting state ensued as the cells reached confluence but could be induced synchronously by trypsinising the cells and inoculating a suspension into Eagle’s medium supplemented with 20-30% calf serum and containing carboxymethyl cellulose (see 6 14.5.1). Although all the cells retained the ability to synthesise collagen (i.e. they are fibroblasts) only a proportion of the cells in a series of different subclones actually produce lipid and this fraction never reaches 100%even after several weeks of incubation of resting cells. The subsequent ability of cells to induce triglyceride synthesis and storage is interfered with by incubation of growing (but not resting) cells with bromodeoxyuridine at 5pM. This compound is known to interfere with several differentiating systems (Rutter et al., 1973) at concentrations which do not affect the rate of cell growth.

15.6. Differentiated hepatocytes The survival of hepatocytes is culture is limited and most cells lose their differentiated functions within 1 or 2 days of culture. Dich et

CH. 15.

DIFFERENTIATION IN CELL CULTURES

309

al., (1988), however, report a medium based on Waymouth’s medium (Dashti et al., 1980) supplemented with albumin, fatty acids and dexamethasone, insulin and glucagon whch allows rat hepatocytes to survive without division for 2-3 weeks. The cells maintained most hepatocyte functions including albumin secretion and urea synthesis.

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CHAPTER 16

Appendices APPENDIX1

Media formulations

TABLE 1 Earle’s and Hanks’ balanced salt solution (BSS) 1OX concentrated, without bicarbonate and glucose

To make 10 litres NaCl KCI MgSO4 7H 2 0 Na 2HP0,. 7H ,O KH2P04 NaH PO4.2H ,O CaCI2.6H2O * Phenol red (1%)- Table 7 Distilled water to

Earle’s

Hanks’

680 g 40 g 20 g

800 g

-

40 g 20 g 9 g

14 g

39.3 g 150 rnl

6 g 27.6 g 150 ml

10 1

10 1

In some cases in Hanks’ BSS half the MgS04.7H,0 is replaced by MgC12.6H20. * This is best dissolved separately and added last with stirring. Add 10 ml chloroform and store at 4O C in polythene containers. CMF (calcium- and magnesium-free BSS) is Earle’s BSS made up without the calcium and magnesium. BSS may be required as a saline solution, for example for washing tissue minces in which case 50 ml of the lox concentrate is diluted with 450 ml distilled water. However, if the BSS is to be used as a basis for a cell culture medium (when further ingredients will be added later) 50 ml of the 10 x concentrate is diluted with 350 or 400 ml distilled water. The dilution should be in 500 ml bottles which should be autoclaved at 15 lb pressure for 20 min. BSS may be stored at room temperature. Before use add bicarbonate (20 ml 5.6% for Earle’s BSS and 3 ml 5.6% for Hanks’ BSS) or Hepes buffer ( 5 ml 1 M) to adjust the pH to 7.4; and glucose or other ingredients as desired. 311

312

CELL CULTURE FOR BIOCHEMISTS

TABLE 2 Dulbecco’s phosphate buffered saline (PBS) PBS solution A

1 litre

10 litres

NaCl KCI Na2HP04 KH,PO, pH 1.2

10.00 g 0.25 R

100.0 g 2.5 g 14.4 g 2.5 g

1.44

s

0.25 g

Bottle in 160 or 400 ml amounts. Autoclave at 15 Ib pressure for 20 min to sterilise. Store at room temperature. PBS solution B CaCI2.2H20

1litre 1.0 g

Bottle in 20 or 50 ml amounts. Autoclave at 15 Ib pressure for 15 d n to sterilise. Store at room temperature. PBS solution C MgC12.6H20

1 litre 1.Og

Bottle in 20 or 50 ml amounts. Autoclave at 15 Ib pressure for 15 min to sterilise. Store at room temperature. To constitute PBS proper add 20 ml each of PBS-B and PBS-C to 160 ml PBS-A. Alternatively, add 50 ml each of PBS-B and PBS-C to 400 ml PBS-A.

TABLE 3 Sodium bicarbonate (5.61)

- available

from most suppliers

- Dissolve 56 g NaHCO, in distilled water. Add 1.5 ml 11 phenol red and make up to 1 1. - Sterilise by filtration using a 0.22 pm membrane filter. - Bottle in 20, 100, and 200 ml amounts. - Cap the bottles very tightly using bottles with rubber Lined metal caps if possible. - Check a sample for bacterial contamination in a) Saboraud fluid medium at 3 l o C for one week (see Appendix 4), b) brain heart infusion broth at 37 C for one week (see Appendix 4). - Store at room temperature.

CH. 16

313

APPENDICES

TABLE 4 Glucose (10%w/v)

- Dissolve 100 g glucose in distilled water. - Make up to 1 1 with distilled water. - Sterilise by filtration. - Bottle in 5 and 10 ml amounts and store at - 20 " C.

TABLE 5 Hepes buffer - available from most suppliers -

Dissolve 47.5 g Hepes in 200 ml distilled water and adjust to pH 8.1 with NaOH. Sterilise by filtration using a 0.22 pm membrane filter and store as aliquots of 5 ml at room temperature. Check for bacterial contamination in a) Saboraud fluid medium at 31 C for one week (Appendix 4), b) brain heart infusion broth at 37 C for one week (Appendix 4).

TABLE 6 Antibiotic solution x 100 - available from most suppliers 10,000,000 u 10 3!

Na benzyl penicillin G (Crystopen) Streptomycin sulphate Distilled water to

11

Sterilise by filtration using a 0.22 pm membrane filter. - Bottle in 5, 20, and 50 ml amounts. - Check for bacterial contamination in a) Saboraud fluid medium at 31" C for one week (Appendix 4), b) brain heart infusion broth at 37 C for one week (Appendix 4). Store at -20°C. -

The penicillin and streptomycin can be obtained from Glaxo Labs. (see Appendix 3). Other antibiotics are available in sterile solution from most suppliers.

TABLE 7 Phenol red 1%(for use as an indicator in BSS and media)

- Dissolve 10 g phenol red in 245 ml of distilled water with 5 ml of 5 N NaOH. - Add 1 M HCI drop by drop till a deep blood red colour is obtained. - Make up to 1 1 with distilled water. - Filter once through Whatman's No. 1 filter paper. - Bottle and store at 4°C.

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CELL CULTURE FOR BIOCHEMISTS

TABLE 8 Eagle’s non-essential amino acids (NEAA)X 100 - available from most suppliers.

g/l L-Alanine L-Asparagine-H,O L-Aspartic acid L-Glutamic acid G1y cin e L-Proline L-Serine

0.89 1.50 1.33 1.47 0.75 1.15 1.05

TABLE9 Lactalbumin hydrolysate Lactalbumin hydrolysate purchased as a dry powder from Nutritional Biochemicals (USA.) is kept in brown sealed bottles. It is dissolved to 5 % in Hanks’ BSS and autoclaved at 115°C for 10 min when it may be kept at room temperature for a month or more. If kept frozen a precipitate forms whch will redissolve on heating in a boiling water bath. For use it is diluted tenfold to 0.5% with growth medium.

TABLE10 Chick embryo extract

- Aseptically remove from the egg 10-day-old chick embryos as described in $ 6.5 and wash them in Hanks’ BSS. Homogenise in a blender for 60 sec at maximum speed in an equal volume of Hanks’ BSS. - Stand at 4°C for 1 h and centrifuge at 35,000 g for 20 min. - Freeze supernatant at -20°C overnight. Then thaw, re-spin and re-freeze in aliquots for storage. Extracts can also be made from individual tissues in a similar manner.

TABLE 11 Tryptose phosphate broth Tryptose phosphate (Difco bacto or Oxoid - Appendix 3) Distilled water to Dispense in 50 ml amounts in 4 oz medical flat bottles. - Autoclave at 15 Ib pressure for 15 min to sterilise. - Keep at 37 ” C for 7 days. - Check each bottle for turbidity before storing at room temperature. -

147.5 g 51

315

APPENDICES

CH. 16

TABLE 12 Eagle’s media formulations (amino acid and vitamin components) BME Amino a c i h (mg/l) L- Arginine-HCI L-Arginine L-Cystine L-Glutamine L-Glycine L-Histidine HCI. H,O L-Histidine L- I soleucine L-Leucine L-Lysine-HCI L-Lysine L-Methionine L-Phenylalanine L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine Vitamins (mg/l) Biotin D-Ca pantothenate Choline chloride Folic acid i-Inositol Nico tinamide Pyridoxal-HC1 Riboflavin Thiamin-HCI

17.4 12.0 292.0 8.0 26.0 26.0 29.2 7.5 16.5 24.0 4.0 18.0 23.5 1.o 1.o 1.o 1.o 2.0 1.o 1 .o 0.1 1.0

MEM

Glasgow MEM

Dulbecco’s MEM

126.0

42.0

84.0

24.0 292.0

24.0 292.0

42.0

21.0

48.0 584.0 30.0 42.0

52.0 52.0 72.5

52.4 52.4 73.1

105.0 105.0 146.0

15.0 33.0

15.0 33.0

48.0 10.0 36.0

46.0

47.6 8.0 36.2 46.8

30.0 66.0 42.0 95.0 16.0 72.0 94.0

1.o 1.0 1.o 2.0 1.o 1.o 0.1 1.o

1.o 1.o 1.o 2.0 1.o 1.0 0.1 1.0

4.0 4.0 4.0 7.2 4.0 4.0 0.4 4.0

The amino acids and vitamins are made up in either Earle’s or Hanks’ BSS. Dulbecco’s modification is made up in Earle’s BSS containing extra bicarbonate (3.7 g/l) and glucose (4.5 g/l) and supplemented with ferric nitrate (0.1 mg Fe(NO,),. 9H *O/I). The formulations are based on those originally prescribed by Eagle (1955a, 1959).

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CELL CULTURE FOR BIOCHEMISTS

TABLE13 Concentrated ( X 10) Eagle’s MEM stock solution - available from most suppliers fglutamine. Commercial suppliers, e.g. Flow Laboratories or Gibco (Appendix 3), sell Eagle’s MEM amino acids as a x 50 stock and the vitamins as 100 stock. The former may be stored at room temperature but the latter should be stored frozen until required. They may be combined aseptically, but as a glucose and glutamine also have to be added and these require filter sterilising, it is more convenient to sterilise after ~ preparation of a 1 0 stock: MEM * amino acids X50 200 ml MEM vitamins XlOO 100 ml L-Glutamine * * 2.925 g Glucose 45 g 11 Glass distilled water to - Adjust the pH to 7.1 using 5 M NaOH (approx. 10 ml). - Sterilise by filtration using a 0.22 pm membrane filter. - Bottle in 50, 100, 200, and 400 ml amounts. - Check for bacterial contamination in a) Saboraud fluid medium at 31° C for one week (Appendix 4), b) brain heart infusion broth at 37 C for one week (Appendix 4). - Store at 4O C until the results of the checks are known.

* To make Glasgow MEM use 200 ml BME amino acids

* * L-Glutamine may be omitted and added to 1X

X 100. medium immediately before use (1

ml of 200 mM solution per 100 ml medium).

TABLE 14 Concentrated ( x 10) Dulbecco’s MEM stock solution - available from most suppliers fglutamine. This is made up similarly to Eagles’ MEM amino acids X 25.

X

10 stock solution by using Dulbecco’s

to make 1 litre Dulbecco’s amino acids X 25 MEM vitamins X 100 Glucose Distilled water pH to 7.1 using 5 M NaOH -

4mz--400 ml 45 g 200 ml

Sterilise by filtration using a 0.22 pm membrane filter. Bottle in 50 and 100 ml amounts. Check for bacterial contamination as above.

N.B. L-Glutamine is added to l x medium immediately before use (10 ml 200 mM glutamine to 500 ml growth medium).

317

APPENDICES

CH. 16

TABLE 15 Preparation of Eagle’s media from concentrated stock solutions The following formulations are based on the use of Eagle’s MEM lox made up without salts (see Table 13) or Dulbecco’s MEM X 10 (see Table 14). However, if the 10 x stock is made with salts (i.e. if it is made from powder) then the BSS should be replaced by sterile glass-distilled water. (a) Eagle’s minimum essential medium supplemented with 10 % calf serum (EC10) (used as growth medium for most routine cell lines, e.g. L929, HeLa, CHO - supplemented with proline for pro- lines - BHK21/C13 and some polyoma transformed lines). BSS * Eagle’s MEM 10 X stock NaHCO, (5.6% - Table 3) Calf serum Antibiotics ( X 100 - Table 6)

450 ml 50 ml 20 ml 50 ml 5ml

(b) Glasgow modification of Eagle’s medium supplemented with 10% calf serum and I0 % tryptose phosphate broth gives improved growth of BHK21/C13 and polyoma transformed lines. 400 ml BSS * 50 ml Glasgow MEM 10 X stock 20 ml NaHCO, (5.6% - Table 3) Calf serum 50 ml 50 ml Tryptose phosphate (Table 11) 5ml Antibiotics ( X 100 - Table 6) (c) Eagle$ minimum essential medium supplemented with 10 % foetal calfsenun and non-essential amino aciak is recommended for growth of BSCI, CVI and PT-K cells and may produce better growth of L929 and HeLa cells. BSS * Eagle’s 10 x stock NaHCO, (5.6% - Table 3) Foetal calf serum NEAA ( x 100 - Table 8) Antibiotics ( X 100 - Table 6)

450 ml 50 ml 20 ml 50 ml 5ml

5ml

(d) Dulbecco’s MEM may be supplemented with 10%calf serum, 20% foetal calf serum or mixtures of the two. BSS * Dulbecco’s x 10 stock NaHCO, (5.6% - Table 3) Foetal calf serum Antibiotics ( X 100 - Table 6) Glutamine (200 mM)

mml 50 ml 30 ml 100 ml 5ml

10 ml

318

CELL CULTURE FOR BIOCHEMISTS

Notes to Table 15 * The BSS in all cases is made up from 50 ml BSS X10 stock diluted to the appropriate volume with distilled water and sterilised by autoclaving (Table 1). N.B. All ingredients should be added aseptically to the bottle of BSS and the medium warmed to 37°C before use. The media may be stored at 4OC for a period of 2-3 weeks. If antibiotics are omitted (the recommended procedure) a sample of the growth medium should be incubated at 37OC for 3 days and at room temperature for a further 2 days and bottles showing contamination discarded. TABLE16A Ham’s medium F12 (from a formulation of Ham, 1965). ma/l

mg/ml

Amino arid

L-Alanine L-Arginine-HC1 L- Asparagine L-Aspartic acid L-Cysteine-HCl.H 2 0 L-Glutamic acid L-Glutamine GIycine L-Isoleucine L-Leucine Vitamins Biotin Ca-pantothenate Choline chloride Folic acid Lipoic acid

3.91 210.7 15.01 13.31 35.12 14.71 146.2 7.51 3.94 13.12 0.007 0.258 13.96 1.324 0.206

L-Lysine-HC1 L-Methionine L-Phenylalanine L-Proline L-Setine L-Threonine L-Tryptophan L-Tyrosine L-Valine

Niacinamide Pyridoxine-HCl Riboflavin Thiamin-HCI Vitamin B12

36.54 4.48 4.96 34.53 10.51 11.91 2.04 5.44 11.71

0.037 0.062 0.038 0.337 1.357

Salts KCI NaCl Na HPO, .7 H 2 0 FeSO,. 7H 2O MgC12.6H20

223.65 7.6 268.1 0.834 122.0

CaCl 2 ’ 2 H 2 0 CUSO,. 5H2O ZnSO, .7 H 2 0 NaHCO,

44.11 0.0025 0.863 1.176

Other components Phenol red Glucose Na-pyruvate Putrescine-2HC1

1.242 1801.6 110.1 0.161

Hypoxanthine Myo-inositol Thymidine Linoleic acid

4.083 18.02 0.727 0.084

CH. 16

319

APPENDICES

TABLE16B Medium MCDB402 (Shipley and Ham, 1981)

%/I

mg/l Amino acids L- Arginine-HCI L-Asparagine.H 2 0 L-Aspartic acid L-Cystine L-Glutamic acid L-Glutamic Glycine L-Histidine. HCI .H 2 0 L-Isoleucine

Vitamins D-Biotin Folinic acid (Ca salt) Lipoic acid Nicotinamide D-Pantothenic acid (hemica salt) Other organic compounds Adenine Choline chloride D-Glucose i-Inositol

63.21 15.01 1.33 48.00 1.47 731.00 7.51 419.20 131.20 0.00733 0.601 0.00206 6.10 11.90

L-Leucine L-Lysine-HCI L-Methionine L-Phenylalanine L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine

262.40 146.10 29.80 49.50 10.51 59.55 2.04 36.24 234.20

Pyridoxine HCI Riboflavin Thiamine-HCI Vitamin B12

20.57 0.376 33.73 0.0135

0.135 13.96 1000.00 7.20

Linoleic acid Putrescine Sodium pyruvate Thymidine

0.0841 0.000161 110.1 0.242

235.00 298.20 197.10

NaCl Na ,HPO,. 7H ,O

Major inorganic salts

CaCl ,.2H20 KCI MgSO, .7H ,O Trace elements CUsod ' 5H2O FeSO, .7H 2O MnC1,.4H20 O"&Mo,O2,.4H

2 0

0.0012 0.278 0.000197 0.0000788

H ,SeO, Na ,Si0,.9H20 NaVO,. 1 4 H 2 0 ZnSO, .7H ,O

6429.50 134.00

0.00129 2.84 0.00187 0.287

Buffers and indicators

NaHCO, Phenol red (Na salt)

1176.20 12.41

5%

N.B. The preparation of medium MCDB402 from stock solutions is described in Shipley and Ham (1981). The pH should be adjusted to pH 7.4 before addition of sodium bicarbonate. The medium will then be at the correct pH in the presence of 5 1 CO,. It should be equilibrated with 5% CO, before use.

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CELL CULTURE FOR BIOCHEMISTS

TABLE 17 McCoy’s 5a medium (RPMI 1629) (originally formulated by McCoy et al., 1985 and modified by Hsu and Kellogg, 1960 and Iwakata and Grace, 1964).

mg/l Amino acids L- Alanine L- Arginine-HCI L- Asparagine L-Aspartic acid L-Cysteine L-Glutamic acid L-Glutamine Glycine L-Histidine-HCI-H,O L- Hydroxyproline Vitamins Ascorbic acid Choline chloride o-Ca-pantothenate i-Inositol Nicotinic acid Pyridoxal-HCI Thiamin-HCI

13.90 42.10 45.00 19.97 31.50 22.10 219.20 7.50 20.96 19.70

0.50 5.00 0.20 36.00 0.50 0.50 0.20

L-Isoleucine L-Leucine L-Lysine-HC1 L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine L-Valine

39.36 39.36 36.50 14.90 16.50 17.30 26.30 17.90 3.10 18.10 17.60

Biotin Folk acid Nicotinamide p-Aminobenzoic acid Pyridoxine-HCI Riboflavin Vitamin B12

0.20 10.00 0.50 1.oo 0.50 0.20 2.00

Salts CaCl MgS04.7H,0 NaHCO,

100.0 200.0 2200.0

KCI NaCl NaH,P04. H,O

Other components Glucose Bacto-peptone

3000.0 600.0

Phenol red Glutathone Foetal calf serum

,

400.0 6460.0 580.0 10.0 0.50 0 -301%

CH. 16

321

APPENDICES

TABLE 18 Medium 199 (As formulated by Morgan et al., 1950,1955).

ma/l Amino acids L-Alanine L- Arginine-HCI L-Cysteine-HC1 DL-Glutaminc acid-H,O Glycine L-Hydroxyproline DL-Leucine DL-Methionine L-Proline DL-Threonine L-Tyrosine Vitamins Ascorbic acid Calciferol Choline chloride i-Inositol Niacin p-Aminobenzoic acid Pyridoxine-HCI Thiamine-HCI Inorganic salts

,

CaCl (anhydrous) Ferric nitrate Fe(N4)3 KC1 KH PO, MgSO4 7H 2 0 NaCl NaHCO, NaH, PO,. H 2O Na HPO, .2H ,O

,

Other components Adenine sulphate Adenylic acid Alpha tocopherol phosphate (sodium salt) Glutathione Hypoxanthine Ribose Thymine Uracil

50.0 70.0 0.1 150.0 50.0 10.0 120.0 30.0 40.0 60.0 40.0 0.050 0.100 0.500 0.050 0.025 0.050

0.025 0.010 Hanks’ salts (mg/l) 140.0

DL-Aspartic acid L-Cystine L-Glutamine r-Histidine-HC1 DL-Isoleucine L-Lysine-HC1 DL-Phenylalanine DL-Serine DL-Twptophan DL-Vdine Biotin Ca-Pantothenate Folk acid Menadione Niacinamide Pyridoxal-HCI Riboflavin Vitamin A (acetate)

60.0 20.0 100.0 20.0 40.0 10.0 50.0 50.0 20.0 50.0 0.010 0.010 0.010 0.010 0.025 0.025 0.010 0.14

Earle’s salts (mg/l) 200.0

0.1

0.1

400.0 60.0

400.0

200.0 8000.0 350.0

-

200.0 6800.0 2200.0 140.0

60.0 10.0

0.20 0.01 0.05 0.30 0.50 0.30 0.30

Adenosinetriphosphate (disodium salt) 1.oo Cholesterol 0.20 0.50 Deoxyribose Glucose 1Ooo.o Guanine-HC1 0.30 Phenol red 20.0 Sodium acetate 50.00 Tween 80c 20.00 Xanthine 0.30

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CELL CULTURE FOR BIOCHEMISTS

TABLE19 Medium NCTC 135 (as formulated by Evans et al., 1964).

Amino acids L-Alanine L-a-Amino-n-butyric acid L-Asparagine-H,O L-Cystine, disodium salt L-Glutamine Glycine L-Hydroxyproline L-Leucine L-Methionine L-Phenylalanine L-Serine L-Threonine L-Tyrosine Vitamins L-Ascorbic acid Choline chloride D-Calcium pantothenate i-lnositol Nicotinic acid Nicotinamide Pyridoxal-HCI Riboflavin m-a-Tocopherol phosphate, disodium salt Salts CaCl,. 2 H 2 0 MgSO4.7H2O NaHCO, Other components Cocarboxylase Deoxyadenosine-H20 Deoxyguanosine-H20 D-Glucosamine-HC1 D-Glucuronolactone NAD Sodium acetate Sodium glucuronate. H,O Thymidine UTP, trisodium salt dihydrate

31.48 5.51 9.19 12.41 135.7 13.51 4.09 20.44 4.44 16.53 10.75 18.93 16.44 50.00 1.25 0.025 0.125 0.0625 0.0625 0.0625 0.025 0.025 264.9 204.8 2200 1.00 10.00 10.00 3.85 1.80 7.00 30.14 1.80 10.00 1.oo

L- Arginine-HCI L-Aspartic acid L-Glutamic acid Glu tathione L-Histidine-HCI. H,O L-Isoleucine L-Lysine-HC1 L-Omithine-HC1 L-Proline Taurine L-Tryptophan L-Valine

Biotin Calciferol Folic acid Menaphthone sodium Bisulphite trihydrate p-Aminobenzoic acid Pyridoxine-HCI Thiamin-HC1 Vitamin A acetate Vitamin B12

31.16 9.91 8.26 10.00 26.65 18.04 38.43 9.41 6.13 4.18 17.5 25.00

0.025 0.25 0.025 0.048 0.125 0.0625 0.025 0.29 10.00

KCl NaCl NaH,. P04.2H20

400.0 6800 158.3

Coenzyme-A Deoxycytidine-HCI FAD, disodium salt Glucose 5-Methylcytosine NADP, sodium salt dihydrate Sodium phenol red Tween 80

2.50 10.00 1.oo 1000 0.10

1.oo 20.00 12.50

CH. 16

323

APPENDICES

TABLE 20 Medium CMRL 1066 (as formulated by Parker et al. 1957).

mg/l Amino acids L- Alanine L- Arginine-HCI L-Cysteine-HCI.H,O L-Glutamic acid Glycine Hydroxy-L-proline L-Leucine L-Methionine L-Proline L-Threonine L-Tyrosine Vitamins p-Aminobenzoic acid Ascorbic acid Ca-pantothenate Cholesterol Folic acid Niacin Riboflavin Inorganic salts CaC1, (anhyd) MgSO,. 7H2O NaHCO,

25.0 70.0 260.0 75.0 50.0 10.0 60.0 15.0 40.0 30.0 '40.0 0.050 50.000 0.010 0.200 0.010 0.250 0.010 200.0 200.0 2200.0

Orher components Cocarboxylase 1 .o 10.0 Deoxyadenosine Deoxyguanosine 10.0 Sodium acetate.3Hq0 83.0 4.2 Sodium glucuronati. H,O Triphosphopyridine nucleotide 1 .oo Ethanol 16.00 Glucose 1Ooo.o Glutathione 10.0 Phenol red 20.0

mg/l

L-Aspartic acid L-Cystine r-Glutamine L-Histidine-HC1.H,O L-Isoleucine L-Lysine-HC1 L-Phenylalanine L-Serine L-Tryptophan L-Valine

Biotin Choline chloride i-Inositol Niacinamide Pyridoxal-HCI Pyridoxine-HC1 Thamin-HCI KCI NaCl NaH, PO,. H,O Coenzyme-A Deoxycytidine Diphosphopyridine nucleotide Thymidine Tween 80 Uridine triphosphate Flavin adenine dinucleotide 5-Methyldeoxycytidine

30.0 20.0 100.0 20.0 20.0 70.0 25.0 25.0 10.0 25.0

0.010 0.500 0.050 0.025 0.025 0.025 0.010 400.0 6799.0 140.0 2.5 10.0

7.0 10.0 5.0 1.o

1 .o 0.1

324

CELL CULTURE FOR BIOCHEMISTS

TABLE 21 Iscove’s medium (Guilbert and Iscove, 1976) mg/l Amino acih L -A1a nine L- Arginine-HC1 L-Asparagine-H,O L-Aspartic acid ~-Cystine-2HCI L-Glutamic acid L-Glutamine Glycine L-Histidine-HCI .H,O L-Isoleucine

25.0 84.0 28.40 30.00 91.24 75.00 584.00 30.00 42.00 105.00

Vitamin Biotin D-Ca pantothenate Choline chloride Folic acid i-I nositol

0.013 4.00 4.00 4.00 7.20

Salts CaCl, (anhydrous) KCI KNO, MgS04.7H,0

165.00 330.00 0.076 200.00

mg/l L-Leucine L-Lysine-HC1 L-Methionine L- Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine (Na salt) L-Valine Nicotinamide Pyridoxine HCI Riboflavin Thiamine HCI Vitamin B12

105.00 146.00 30.00 66.00 40.00 42.00 95.00 16.00 104.20 94.00 4.00 4.00 0.40 4.00 0.013

NaCl NaHCO, NaH,P04.2H,0 Na ,Se03. 5H,O

4505.00 3024.00 141.30 0.0173

Phenol red Sodium pyruvate

15.00 110.00

Supplements for Costar’s SF-1 medium (Cleveland el al., 1983) Bovine serum albumin 2500.00 Linoleic acid 2.00 (pl) Palmitic acid Ethanolamine 5.00 Oleic acid Insulin 36.00 Trans ferrin

1.oo 1.OO 1.00

Other additives Glucose Hepes

4500.00 5962.00

325

APPENDICES

CH. 16

TABLE 22 Gamborg B5 medium (Gamborg et al., 1968) - available in powder form from most suppliers.

Salts NaH2P04 KNO, (NH 4 1* so, MgSO,. 7H2O CaCl ,.2H20 EDTA (Fe salt) MnSO,. H 2 0 Vitamins Nicotinic acid Thiamin HCI Pyridoxin HCI

150.00 3000.00 134.00 500.00 150.00 28.00 10.00

H3BO3 ZnSO,. 7H 2O Na ,MOO,. 2 H 2 0 CUSO, CoCl2’6H20 KI

Sucrose 2 4-D m-Inositol

1.00 10.00 1.oo

3.00 2.00 0.25 0.025 0.025 0.75

20000.00 2.00 100.00

This should be prepared as a 10%concentrate when the final medium can be prepared using: 100 ml lox stock

200 ml deproteinised coconut milk

*

700 ml H 2 0

The final pH should be approximately 5.5. * This is prepared by autoclaving coconut milk and filtering through a Whatman NO. 1 filter. It can be resterilised by autoclaving.

APPENDIX 2 Stains and fixatives

A number of stains and fixatives have been referred to throughout the book and these can be located by using the index. Here are listed some of the more common ones. (1) Acetic orcein - Fix cells in methanol :acetic acid (3 : 1) and air-dry. - Stain for 3-5 min with 2% orcein in 4% acetic acid. - Rinse in 40% methanol and air-dry.

(2) Acridine orange fluoresence stain To a a suspension of cells in BSS is added a drop of an aqueous 0.01% solution of acridine orange. On examination under the fluorescent microscope nuclei appear yellowish green and cytoplasm red.

326

CELL CULTURE FOR BIOCHEMISTS

(3) Carnoy’s fixatioe 1 part glacial acetic acid 3 parts absolute ethanol (4) Formal saline

- Dissolve 5 g NaCl and 15 g Na,SO., in distilled water and make up to 900 ml. - Filter 40% formaldehyde using Green’s filter paper No. 9044 and add 100 ml to the salt solution. ( 5 ) Giemsa stain - To 30 g Giemsa add 1980 ml glycerol. - Heat at 56OC for 90-120 min. - Add 1980 ml methanol and mix well. - Stand at room temperature for 7 days and filter through Green’s 904; filter paper. For w e : Dilute 1/10 in buffered distilled water or use undiluted.

(6) Gram srain a) Methyl violet 6B 8g Absolute ethanol 80 ml 1% aqueous ammonium oxalate 320 ml b) Iodine Ig Potassium iodide 2g Distilled water 100 ml c) Safranin 2.5 g Ethanol 100 ml Distilled water to 11 - Pour methyl violet solution (a) onto the fixed preparation and remove after 30 sec. - Wash in tap water. - Pour on iodine solution (b) and remove after 1 min. - Wash with water and decolorise with ethanol until no more stain is removed. - Apply safranin counter stain solution (c) for 2 min. - Wash with water and dry at room temperature. - Examine under oil immersion. ( 7 ) Haematoxylin and eosin The haematoxylin is dissolved in 95%ethanol and to 6 ml of a 17% solution is added 100 ml saturated ammonium alum solution and 0.5 g mercuric oxide. After boiling and cooling 25 ml of glycerol and 25 ml of methanol are added and the whole mixture filtered.

- Stain the fixed cells overnight in a 100-fold dilution of haematoxylin mixture. Rinse in water for 15 min. - Counter stain in 0.5% eosin Y for 0.5-1 min and rinse.

-

Nuclei are stained blue and cytoplasm red.

CH. 16

APPENDICES

327

(8) Leishrnan stain - To 7.5 g Leishman stain add 5 I methanol and shake at intervals over 5 days before use. (9) May-Grunwald Giemsa stain - Dilute 9 ml stock Giemsa (0.3%in glycerol methanol) with 90 ml 0.1 M phosphate buffer pH 6.8 and add 1 ml of May-Grunwald stain (0.25%w/v in metahnol).

(10) Methylene blue The stain is 30 ml saturated ethanolic solution of methylene blue mixed with 100 ml 0.01% aqueous KOH. - Stain the preparation for 3 min and wash with water. - Blot dry and examine microscopically under oil immersion.

This stain is used so that all bacteria are more easily seen under high power objectives. (1 1) Orcein stain

A 2% solution of natural orcein (Gurr’s) in 60% glacial acetic acid is prepared by dissolving the orcein in boiling glacial acetic acid with stirring. Cool to 55OC. Add distilled water to make the acid 60%.Cool to room temperature. Filter twice through Whatman filter paper No. 1. (12) Hoesch fluorescence stain - Wash cells in PBS and fix in ethanol: acetic acid (1 : 1) for 2 min. - Wash twice in water and stain with a solution of Hoesch 33342 (1.3 pg/ml H,O) for 1 h at 37OC in the dark (cover the dish with aluminium foil). - Wash twice in water, dry and mount in buffered glycerol. - Examine under the fluorescence microscope when DNA stains greenish/yellow.

APPENDIX 3 The following suppliers are those with whom 1 have had contact. The list is not exclusive, nor does the presence of the name of a supplier imply recommendation of their products. The suppliers, or their agents, are largely British and in some instances there is a Scottish bias and this again is largely a result of my experience. Further information may be obtained from the Laboratory Equipment Directory published annually by Morgan-Grampian Book Publishing Company Limited, 48 Beresford St., London SE18 6BQ; or from International Laboratory, 30 Controls Drive, P.O. Box 870, Shelton, CT 06484-0870, U.S.A. Suppliers

Abbot Laboratories, Diagnostic Div., Moorbridge Rd., Maidenhead, Berks. American Type Culture Collection, Rockville, M D 20852, U S A . Amicon Ltd., Upper Mill, Stonehouse, GB-Glos GLlO 2BJ. Anderman & Co., Ltd., London Rd., Kingston-upon-Thames, Surrey, KT2 6NH.

328

CELL CULTURE FOR BIOCHEMISTS

Charles Austen Pumps Ltd., 100 Royston Rd., Byfleet, Surrey KT14 7PB. Beckman Instruments, Progress Rd., Sands Industrial Est. High Wycombe, Bucks. HP12 3BR or P.O. Box 10200, Palo Alto, CA 94304, U.S.A. Becton Dickinson U.K. Ltd., Between Towns Rd., Cowley, Oxford OX4 3LY. Becton-Dickinson, 2375 Gaveia Ave., Mountain View, CA 94043, U.S.A. Bellco Glass Inc., P.O. Box B, 340 Edrudo Rd., Vineland, NJ 08360, U.S.A. (see A.R. Horwell). Beveridge, A. & J., 5 Bonnington Rd. Lane, Edinburgh. Bibby Science Products Ltd., Stone, Staffordshire ST15 OSA. Bio-Rad Laboratories Ltd., Maylands Ave., Hemel Hempstead, Herts. HP2 7TD. Bioassay Systems (LH Fermentation Ltd.,), Bells Hill, Stoke Poges, Slough, Bucks. SL 2 4EG. Boehringer Co. Ltd., Bell Lane, Lewes, E. Sussex BNT IBR. British Drug Houses Ltd., Poole. Dorset BH12 4”. British Oxygen Co. Ltd., 24 Deer Park Rd., London, or 150 Polmadie Rd., Glasgow G5. Browne, A. Ltd. (see R. & J. Wood Ltd.). CC Laboratories, Dorcas Lane, Stoke Hammond, Milton Keynes, MK 17 OEA (see Mediatech, Inc.). Calbiochem Ltd. (Novabiochem U.K. Ltd.) 3 Heathcoat Building, Highfields Science Park, Nottingham NG7 245. Camlab Ltd., Nuffield Rd., Cambridge CB4 1TH. Cellon Sarl, 22 rue Dernier-Sol, L-2543 Luxembourg. Connaught Laboratories (see A.R. Horwell Ltd.). Corning Ltd. (see Bibby Science Products Ltd.). Costar Ltd. (see Northumbria Biologicals Ltd.). Coulter Electronics Ltd., Northwell Dr., Luton, Beds. LU3 3RH. Cryoson. Benelux Bv., Postbus 15, 146226, Middenbeemster, Holland. Damon/IEC (U.K.) Ltd., Unit 7, Lawrence Way, Brewster Hill Rd., Dunstable, Beds. LU6 1BD. Decon Laboratories Lid.. Conway St., Hove, E. Sussex BN3 3LY. Difco Laboratories, P.O. Box 14B, Central Ave., East Molesey, Surrey KT8 OSE. Difco Laboratories, P.O. Box 1058, Detroit, MI 48232, U.S.A. Distillers Co., Ltd., Cedar House, 39 London Rd., Reigate, Surrey RH2 9QE. Dow Coming Corp., Box 0994, Midland, MI 48686-0994, U.S.A. (see McFarlane Robson). Du Pont (U.K.) Ltd., Wedgewood Way, Stevenage, Herts, SG1 4QN. Dynal (U.K.) Ltd., Station House, 26 Grove St., Wirral, Merseyside L62 2AB. Dynatech Labs Ltd. (see Gibco). Elga Group, Lane End, High Wycombe, Bucks. HP14 3JH. Epsom Glass Industries Ltd., Longmead, Epsom, Surrey KT19 9RN. European Collection of Animal Cell Cultures, PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury SP4 OSG. Evans Medical Supplies Ltd., Ruislip, London. Finnpipettes (see Labsystems, U.K., Ltd.). Fisons Scientific Apparatus Ltd., Loughborough, Leics. LEI 1 ORG. Flow Laboratories, Woodcock Hill, Harefield Rd., Rickmansworth, Herts. WD3 1PQ. Forma Scientific - Mallinckrodt Inc., Millcreek Rd., P.O. Box 649, Marietta OH 46750, U.S.A. - Raven Scientific Ltd., P.O. Box 2, Haverhill, Suffolk.

CH. 16

APPENDICES

329

Gallenkamp Ltd., Belton Road West, Loughborough LEll OTR. Gelaire (see Flow Laboratories). Gelman Sciences Ltd., 10 Harrowden Rd.. Brockmills, Northampton NN4 OEZ. Gene-Probe Inc., 9620 Chesapeake Dr., San Diego, CA 92123, U.S.A. (see Lab. Impex Ltd.). Gibco, Ltd.. P.O. Box 35, Trident House, Paisley, Scotland, and 3175 Staley Rd., Grand Island, N.Y., 14072, U.S.A. Glaxo Laboratories Ltd., Greenford, Middlesex. Grant Instruments Ltd., Barrington, Cambridge CB2 5QZ. Green’s Filter Paper (see Whatman Lab. Sales Ltd.). Gurr, G.T. Ltd. (see British Drug Houses). Heraeus GmbH-, P.O. Box 1553, D-6450 Hanau, West Germany, and Unit 9, Wates Way, Brentwood, Essex CM15 9TB. Hoechst (U.K.) Ltd., Hoechst House, Salisbury Rd., Hounslow, Middx TW 4 6JH. Hopkins and Williams (see British Drug Houses). Honvell, A.R., Ltd., 73 Maygrove Rd., West Hampstead, London NW6 2BP. Hotpack - 10940, Dulton Rd., PA 19154, U.S.A. (see Daman, IEC). Howe, V.A. & Co. Ltd., 12-14 St Ann’s Cresc., London SW18 2LS. Human Genetic Mutant Cell Repository, Inst. for Medical Research, Copewood St., Camden, NJ 08103, U.S.A. Hyflo Pumps (see Metcalf Bros Ltd.). ICN Biomedicals Ltd., Lincoln Rd., Cressex Ind. Estate, High Wycombe, Bucks. HP12 3XJ, or Free Press House. Castle St., High Wycombe, Bucks. HP13 6RN. Ilford Ltd. - Colour Lab. Suppliers, 110 Toryglen St., Shawfield, Glasgow. Imperial Laboratories Ltd., Ashley Rd., Salisbury SP2 7DD. International Biotechnologies Ltd., Kiryat Hadassah, P.O. B-12000, Jerusalem 91120, Israel. Jencons Ltd., Cherrycourt Way Ind. Est., Stanbridge Rd., Leighton Buzzard, Beds. LU7 8UA. KC Biologicals (see Sterilin). Koch-Light Ltd., Edison House, 163 Dixons Hill Rd., North Mymms, Hartfield, Herts. AL9 7JE. Kodak Ltd., P.O. Box 66, Kodak House, Station Rd., Hemel Hempstead HPI IJU. Kontes Biotechnology, 1022 Spruce St., Vineland, NJ 08360-2899 (see also Hoefer Scientific Instruments U.K., P.O. Box 351, Newcastle, Staffs, ST5 Om). LEEC, Private Rd., No. 7, Colwick Estates, Nottingham NG4 2AJ. Lab Impex Ltd., 111-113 Waldegrove Rd., Teddington, Middlesex Twll 8BR. Lab-Tek (see Nunc Inc. or ICN Biomedicals Ltd.). Labmart Ltd., 1 Pembroke Ave.. Waterbeach, Cambridge CB5 9QR. Labsystems (U.K.) Ltd., 12 Bedford Way, Uxbridge, Middlesex UB8 1SZ. Laser Lab-Systems Ltd., P.O. Box 166, Southampton SO9 7LP. Linbro (see Flow Laboratories). Lorne Laboratories Ltd., P.O. Box 6, Twyford, Reading, Berks. RGlO 9NL. Luckham Ltd., Labro Works, Victoria Gdns., Burgess Hill, Sussex, RH15 9QN. Lux (see ICN Bromedicall Ltd.). MDH Ltd., Walworth Rd., Andover, Hants. SPlO SAA. May & Baker Ltd., Liverpool Rd.. Eccles, Manchester M30 7RT. McFarlane Robson Ltd. (see British Drug Houses). McQuilken & Co., 21 Polmadie Ave., Glasgow G5.

330

CELL CULTURE FOR BIOCHEMISTS

Mediatech Inc., 13884 Park Center Rd., Hernden, VA 22071, U.S.A. (see CC Laboratories). Medilog bv., Marijkelaan 11, Postbus 95, 2420 AB Niewkoop, Holland. Metcalf Bros. Ltd., Cranbourne Rd., Potters Bar, Herts. Millipore (U.K.) Ltd., The Boulevard, Ascot Rd., Croxley Green, Watford WDl 8WD. Millipore Corp., 80 Ashby Rd., Bedford, MA 01730, U.S.A. Moredun Animal Health Ltd., 408 Gilmerton Rd., Edinburgh EH17 7JH. Napco Scientific Co., P.O. Box 1000, 20210 S.W. Teton, Tualatin, OR 97062-1000, U.S.A. National Diagnostics, Unit 3, Chamberlain Rd., Aylesbury, Bucks. HP17 3DY, and 1013-1017 Kennedy Blvd., Manville, NJ 08835, U.S.A. New Brunswick Scientific (U.K.) Ltd., 6 Colonial Way, Watford, Herts. WD2 4PT (see also Koch-Light Ltd.). Nikon (U.K.) Ltd., Haybrook, Halesfield 9, Telford, Shropshire, TF7 4EW (see also Gallenkamp). Northumbria Biologicals Ltd., South Nelson Ind. Est., Cramlington, Northumberland NE23 9HL. Nunc-Inc., 2000 North Aurora Rd., Naperville, Illinois, IL 60566, U.S.A. (see also Gibco). Nutritional Biochem C o p . (see Uniscience Ltd.). Olympus Microscopes (see Gallenkamp). Organon Ltd., Science Park, Milton Rd., Cambridge CB4 4FL. Oxoid Ltd., Wade Rd., Basingstoke, Hants. RG24 OPW. Permutit Co. Ltd., 632/652 London Rd., Isleworth, Middlesex TW7 4EZ. Pharmacia (G.B.) Ltd., Pharmacia House, 351 Midsummer Blv. Milton Keynes MK9 3HP. Planer-Biomed, Windmill Rd., Sunbury-on-Thames, Middlesex TW16 7HD. Prior Scientific Instruments Ltd., London Rd., Bishop’s Startford, Herts. CM23 5NB. Queue Systems (see Camlab.). Quickfit & Quartz Ltd. (see Gallenkamp). Raven Scientific Ltd., P.O. Box 2, Haverhill, Suffolk. Repelcote (see Hopkins and Williams). Sarstedt Ltd., 58 Boston Rd., Beaumont Leys., Leicester LE4 IAW. Sartorious Instruments Ltd., 18 Avenue Rd., Belmont SM2 6JD. Schleicher & Schuell Ltd. (see Anderman & Co. Ltd.). Sigma Chem Co. Ltd., Fancy Rd., Poole, Dorset BH17 7NH. Statebourne Cryogenics, 18 Parsons Rd., Washington, Tyne & Wear NE37 1EZ. Sterilin Ltd., Clockhouse Lane, Feltham, Middlesex TW14 80s. Taylor-Wharton (see Jencons Ltd.). Techmation Ltd., 58 Edgeware Way, Edgeware, Middlesex HA8 8JP. Techne Ltd., Duxford, Cambridge CB2 4PZ, and 3700 Brunswick Pike, Princeton, NJ 08540-6192, U.S.A. Thermolyne, 2555 Kerper Blvd., Dubuque, LA 52001, U.S.A. (see Labmart). Tissue Culture Services Ltd., Unit 2, Perth Est., Perth Ave., Slough, Bucks. SL1 4XX. Titertek (see Flow Laboratories). Union Carbide (U.K.) Ltd. (see British Oxygen Co.). Uniscience Ltd., 12-14 St. Anne’s Cresc., Wandsworth, London SW18 2LS. VirTis Co., Inc., Gardner. New York, NY 12525, U.S.A. (see Techmation Ltd.).

CH. 16

APPENDICES

331

Voss Ltd., Victoria Gdns., Burgess Hill, West Sussex RH15 9QN. Whatman Ltd., Springfield Mill, Maidstone, Kent ME14 2LE. Wheaton Scientific (see Jencons Ltd.). Wild Heerbrugg Ltd., CH-9435 Heerbrugg, Switzerland. Wood, R. & J., Ltd., 39 Back Sneddon St., Paisley PA3 2DE. Worthington Biochemical Corp. (see Lome Laboratories). APPENDIX 4 Sterility checks (1) Beef heart infusion broth - Add 50 g beef heart infusion (Difco Labs., Detroit) to 900 ml distilled water at 50 C. Stand for 1 h. - Bring slowly to the boil. - Filter through a double layer of Whatman No. 12 filter paper. - Distribute into bottles and sterilise by autoclaving for 15 min at 15 Ib pressure.

(2) Brain heart infusion broth - Dissolve 37.0 g of brain heart infusion broth powder (Oxoid, Basingstoke) in 1 1 of distilled water. - Bottle in 25 ml and 40 ml amounts in 2 oz medical flat bottles. - Autoclave at 15 Ib pressure for 15 min to sterilise. - Store at room temperature. (3) Sabouraud fluid medium - Dissolve 30 g of Saboraud fluid medium base (Oxoid, Appendix 3) in 1 1 of distilled water. - Bottle in 25 ml and 40 ml amounts in 2 oz medical flat bottles. - Autoclave at 15 Ib pressure for 15 min to sterilise. - Store at room temperature. (4) Blood agar plate - Dissolve 40 g blood agar base (Oxoid, Basingstoke) in 1 I of distilled water by standing in a boiling water bath. - Aliquot in 50 ml parts and autoclave at 121OC for 15 min. - To 50 ml agar base (heated to melt and cooled to 45OC) add 5 ml horse blood - Pour 5 ml into 5 cm Petri dishes and allow to cool. The horse blood can be delivered regularly, e.g. every 2 weeks, from the suppliers (Oxoid Ltd., Basingstoke).

( 5 ) PPLO Agar (A) PPLO agar base - To make 1 I PPLO Agar base mix 34.0 g bacto-PPLO Agar (Difco Labs.) with 1 I cold distilled water in a flask and stand tlus in a bucket of boiling water until the agar dissolves.

332

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CELL CULTURE FOR BIOCHEMISTS

While molten, dispense in 25 ml amounts in 4 oz medical flat bottles. Autoclave at 15 Ib pressure for 15 min to sterilise. Store at room temperature. ml (for 10 plates) 25 7.5 3.75 3.75 0.4 0.15

(B) PPLO agar plates PPLO agar base Horse serum Yeast extract (see below) Tryptose phosphate broth (Difco) Thallous acetate (1.25%) Penicillin

Boil the agar (in a water bath) until molten and hold at 45 " C. Heat the serum and yeast extract to 45°C. - Add the horse serum, yeast extract, tryptose phosphate broth, thallous acetate, and penicillin to the agar. - Mix well and dispense 4 ml amounts into 50 mm Petri dishes. - Allow to solidify and store at 4 " C. -

(C) Yeast extract - Add 250 g dried baker's yeast to 1 1 distilled water and bring to the boil - Filter through a double layer of Whatman No. 12 filter paper. - Add NaOH to raise the pH to 8.0 and dispense into 10 ml aliquots, autoclave and store at - 20 O C. ( 6 ) Soy peptone-yeast dialysate medium (Kenny. 1973).

(A) Basic broth Soy peptone (Sheffield Chem., Co., Nonvich, NY) NaCl H2 0

30 g 5g 11

Adjust to pH 7.4 and add 10 g of Difco Noble Agar if a basic agar is required. Autoclave and store and room temperature. (B) Yeast dialysate 450 g Fleischmann's active dried yeast is added to 1250 ml H,O and autoclaved for 5 min at 121OC. It is then dialysed against 1 1 of water at 4OC for 2 days. The dialysate is autoclaved and stored frozen. (C) Complete medium Basic broth (or agar) Yeast dialysate Horse serum Penicillin Thallium acetate (3.3%)

65 ml 10 ml 25 ml 20,000 u 1 ml

Inclusion of arginine (16 mM) and 0.4 mg% phenol red indicates the presence of arginine deiminase by formation of alkali (purple colouration).

CH. 16

APPENDICES

333

APPENDIX 5 Assays

(1) DNA, RNA and protein estimaiions Using the following procedure DNA, RNA and protein may all be conveniently assayed on the same batch of cells. a) Harvest cells by trypsination and count them using a haemocytometer (3 8.2) or electronic cell counter (5 8.2.2). b) Wash the cell suspension twice in PBS to remove extraneous protein followed by two washes in 5% trichloroacetic acid to remove acid soluble material. c) Dissolve about lo7cells in 1.0 ml 0.10 M NaOH (heat to 40 ” C if necessary). d) Take 0.15 ml solution plus 0.85 ml 0.7 M NaOH. Use for protein estimations (see below). e) To the remaining 0.85 ml solution add 0.15 ml 3.3 M PCA. Incubate at 70°C for 30 min. Centrifuge and retain the supernatant (hot acid extract) for estimation of DNA and RNA. (A) DNA estimniion The diphenylamine reagent is made up according to Burton (1956). To 500 ml glacial acetic acid add 7.5 ml concentrated sulphuric acid and 7.5 g diphenylamine. Mix cautiously and store in a brown bottle at room temperature. Just before use add 0.1 ml 1.6%acetaldehyde to 20 ml of the above mixture. - Place 0.4 ml aliquots of the hot acid extracts (from 1 (e) above) in tubes and make up to 1.0 ml with 0.5 M perchloric acid. - Add 2.0 ml diphenylamine reagent and mix. - Cover and stand at 30°C for 18-48 h and read the absorbance at 600 nm. A standard DNA solution can be made by dissolving native DNA in 50 mM KCI and from its spectrum its concentration may be calculated (Hirschman and Felsenfeld, 1966). Calf thymus DNA dissolved at 1 mg/ml has an E,,, of 18.5. The standard DNA solution should then be made 0.5 M with respect to perchloric acid and heated at 70°C for 30 min to dissolve the DNA. This solution should be diluted to 100 pg/ml with 0.5 M perchloric acid and various amounts up to 1.0 ml used as described above to prepare a standard curve. (B) RNA estimation A standard RNA solution is prepared by dissolving RNA in 0.05 M NaOH at 50 PB/ml. - Place 0.2 ml aliquots of the hot acid extracts from 1 (e) above in large test tubes ( 6 x 5 / 8 ” ) and dilute to 1.5 rnl with water (for the standard curve use up to 1.5 ml of the standard RNA solution). - Add 1.5 ml of 0.03% FeCI, in concentrated HCI and 0.1 ml 20% freshly prepared orcinol in 95% ethanol. - Mix thoroughly and place in a uigorously boiling water bath for 30 min. Use glass ‘dew-drops’ to prevent loss of volume. - Cool in ice-water slurry and read the absorbance at 665 nm.

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(C) Protein estimation Prepare a solution of Coomassie Blue as follows (Bradford, 1976): - Dissolve 100 mg Coomassie Brilliant Blue G in 50 ml 95% ethanol. - Add 100 ml 85% (w/v) phosphoric acid. - Dilute to 1 1 with water and filter. - Take 100 pl samples for protein estimation and add 3 ml reagent. Mix well and leave for 4 min. Read A,,, withn one hour. A standard curve can be made using BSA (5-40 p g ) . (2) Fluorescent DNA assays The diphenylamine method can be scaled down to increase sensitivity and fluorescent methods are available using diaminobenzoic acid, diamidino-phenylindole (DAPI), ethidium bromide or propidium iodide (Kissane and Robins, 1958; Hinegardner, 1971; Klotz and Zimm, 1972) which are sensitive down to 0.1 pg DNA. The flurorescent assay has been developed for use in microtitre plates using DAPI or Hoechst 33342 and is sensitive down to 500 cells/well (McCaffrey et al., 1988). The following simplified procedure is in use in our department (F. Rinaldi, personal communication) for assaying the DNA content of cells growing in 24-well plates. 1) Wash the cells in PBS-A (Appendix 1). 2) Dissolve in 100 pl of 0.2% SDS in ETN (10 mM EDTA; 10 mM Tris.HC1; 100 mM NaCl pH 7.0. 3) Incubate at 37"C, 15 min. 4) Mix with 2.4 ml ETN containing 100 ng/ml Hoechst 33258 plus RNase (5 %/mu. 5) Wrap in foil and incubate at 37°C. 15 min. 6) Measure enhancement at 450 nm in a fluorescence spectrophotometer with an excitation of 360 nm (slit width: 20 nm). (3) Methylene blue cell nucleic acid assay (Pelletier et al., 1988). This is a method of measuring total nucleic acid which is equivalent to cell number. The method described is for use with cells growing in microtitre plates but it can readily be scaled up. - Wash the cells in the wells three times with PBS. - Stand in 10%formalin in PBS for 10 min. - Wash three times in 10 mM borate buffer pH 8.4. - Stain in 1%(w/v) methylene blue in borate buffer for 10 min. - Wash in borate buffer until all excess dye is removed. - Dry and add 100 pl 0.1 N HCI to each well. - Read in a microtitre plate reader (or remove to cuvette) at 660 nm. The washings can be done by immersing the plate in a bath of buffer (ensure no air bubbles remain) and banging the plate dry on a pad of filter paper. The dye should be added and removed using an 8-place dispenser.

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Subject index A-state 193

Antiseptics 168 Arginine deaminase 177 Ascites 113 Aseptic technique 169 Atebrin 140 Attachment factors 19, 22 Attenuated virus 296 Autoclaving 154 Autocrine response 23 Autoradiography 223 of acid soluble material 257 in dishes 255 double label 251 for microplasmas 178 resolution 250 Auxotrophic mutants 263 Azacytidine 265 Azaguanine 265

A9 cells 20

Accumulation functions 203 Acetic orcein 325 Acid soluble autoradiography 257 pool 240 Acridine orange 325 Adenyl cyclase 29 Adhesion 22 Adipocytes 308 Ageing 14 Air lift systems 55 Airborne contamination 168 Albumin 32 Alginate beads 55, 58 Alignment 11 American Type Culture Collection 16 Amethopterin 229, 245, 265 Amino acid deficiency 250 Aminopterin 229, 236, 245, 265 Amplification 265 Ampoules 129 Anchorage dependence 19 Aneuploidy 14, 136 Antibiotic stock 313 Antibiotics 79, 81, 165, 173 Antibody production 292 staining 294 Antifoam reagents 56 Antifolates 229 resistance to 265

B-phase 193 Bacterial contamination 165, 168 Bactopeptone 71 Balanced salt solution (BSS)72 Basement membrane 13 Beef heart infusion broth preparation of 331 BAK 21 cells 15 Bicarbonate buffer 35, 73, 312 Bio-Carriers 52 Biofreeze vials 129 Biopsies 8 Bioreactors 56 Blastogenesis 101 355

356

CELL CULTURE FOR BIOCHEMISTS

Blockade of S-phase 227 Blocking endogenous pathways 245 blood agar plate 167 preparation 71, 331 BME 72 Brain, heart infusion broth preparation 331 Brain/heart infusion 167 BRL cells 93 Bromodeoxyuridine 222 antibody 210 labelling 259 Brown’s tubes 155 BSA 87 BSCl cells 76 Butyrate and transfection 143 Calcium ionophore 28 phosphate, for transfection 142 Callus cultures 95 Canes 132 Capillary beds 46 Carnoy’s fixative 326 3T3 Cells 15, 69 contact inhibition 20 differentiation 224 Cell banks 135 communication 275 cycle 18, 187 control 191 cycle analysis 200 entrapment 55 factory 44 growth test, for micoplasmas 182 line 14 size selection 215 strain 14 stocks, organisation of 135 Centrifugal elutriation 219 Storage of 127, 132

Chamber slide 179 Chemostat 70 Chick embryo cells 105 extract 79, 314 Chick liver cells 106 Chick serum 307 Chloros 151 CHO cells 226 Chromosome 137 sorting 141 Ciprofloxacin 184 Cloning 14, 117, 119 cylinders 119 under agar 120 with a feeder layer 121 CMF 311 CMRL media 79 CO, as essental nutrient 117 concentration 35, 37 incubators 36 Coconut milk 94 Colcemid 137, 203, 213 Collagen 11, 13, 17 beads 54 Collagenase 60 Concentrated stocks 316 Conditioned media 17, 92 Confluence 20 Conjugation of antisera 293 Contact inhibition 15, 20 Contaminated material disposal 174 Contamination 165 of cell lines 16 Coulter counter 124, 127 Counting 122, 126 Coverslip cultures 38 washing 39 CPSR 92 Cryoboxes 132

SUBJECT INDEX

Cryogenic work station 129 Cryptic virus 185 Crystal violet 183 CSF 24, 26, 28 Culture density 11 vessels 35 advantages of 2 cost of 2 experimental system 2 extent of use 4, 10 products of 8 CY1 cells 76 Cycle time 199 Cyclins 195 Cytodex 51, 54 Cytopathic effect 184, 279 Cytosine modification 265 Cytotoxicity tests 7 DAPI staining for micoplasmas 180 Dead cells estimation 210 Death of cells 14 Decon 152 Defined media 88 Deionised water 152 Density dependence 20 Deoxycholate plate 167 DePex 200 Detection of virus 288 7X Detergent 152 Differentiation 4, 32, 301 effect of media supplements 89 Dihydrofolate reductase 229, 265 Dimethylchlorosilane 65 Dipteran cells 114 Disinfectant 168 Dispase 61 Disposable filters 159

357

Dissociation of tissue 59 Distribution of cells around cycle 196 DMSO 128, 302 DNA assay, fluorescent 334 assay (Burton) 333 fibre autoradiography 191 fingerprinting 16 probe, for micoplasmas 183 repair 259 synthesis, rate of 239 Dome formation 13 DonC cells 222 DOTMA 144 Double block technique 229 Double thymidine block 236 Doubling time 198 dTTP synthesis endogenous 242 Dulbecco’s MEM 72 PBS 312 Eagle’s media 75 basal 17 formulations 315 preparation from stocks 317 Earle’s BSS 73, 311 EDTA 61 EGF 24, 87 as progression factor 225 Electrofusion 145, 274 Electronic cell counter 123 Electroporation 144, 274 Elutriation 219 Embryo extract 71 Embryoid bodies 306 Embryonal carcinoma (EC) cells 305 EMC virus 283 EMS 263 Emulsion autoradiographic 251 Endocrine response 23

358

CELL CULTURE FOR BIOCHEMISTS

Endogenous pools allowance for 246 Endogenous synthesis of dTTP blocking 245 Endothelial cells 13, 111 Enzyme-linked antibody 292 Epithelial cells 11, 39 mammary 111 colonic 112 Erythroid differentiation 301 Erythropcetin 24, 27 Exponential growth 18

Fluorescent antibody 292 Fluorodeoxyuridine 232 Foetal bovine serum (FBS) 82 Folic acid 230 Formal saline 326 Freezer liquid nitrogen 128, 132 programmable Freezing 127 procedure 129 in serum-free medium 90 Friend cells 221, 301 Fusion 145, 270, 273

F9 cells 306 FACS 207 FACScan 221 FDGF 24 Feedback inhibition by dTTP 241 Feeder layer 117, 304, 306 Fibrinogen clots 16 Fibroblast overgrowth 85, 87 removal of 32, 33 Fibroblast-like 11 Fibronectin 21, 55, 88 Ficoll-Paque 99 Filters disposable 159 tangential flow 163 Filtration 156 apparatus 157 bell 158 FITC 293 celite 293 Flaming 169 Flattening 22 Flooding the pool of dTTP 243 Flow cytometry 141 Flow microfluorometry 207 Fluorescence microscopy 148 staining 179

G-banding 140 G-proteins 29 GO-phase 191 G1-phase duration 192 Gamborg B5 medium formulation 325 Gap junctions 276 Gelatin 13 -coated dishes 19, 41 Geli-beads 54 Generation number 16 time 199 Genetics 4 Germicidal lamp 169 Giant cells 18 Giemsa 140, 326 Glasgow MEM 72 Glass bead columns 46 Glass-coated beads 52 Glassware preparation 151 Globulin preparation 293 Glucose solution 313 Glutamine stability 77 Glycerol for freezing cells 127 Glycerol mountant 179 Grain counting 255

SUBJECT INDEX

Gram stain procedure 326 Graphical analysis of cell cycle 206 Growth control 16 cycle 68, 70 factors 23 fraction 197, 198 Haemadsorption 295 Haemagglutination 295 Haematoxylin and eosin 326 Haemocytometer 122 Haemopoietic cells 13, 26 Ham’s F12 formulation 318 Ham’s media 78 Hamster cells 225 Hanks’ BSS 73, 311 HAT selection 265, 270 Hayflick phenomenon 14 HECS 90.93 HeLa cells 15 nutritional requirements 17 HeLa S3 cells 20 HEP2 cells 15 HEPA filter 168, 172 Hepatocytes 107, 308 Hepes buffer 35, 75, 313 Herpes simplex virus 283 Hirt extraction 285 Histopaque 99 HMBA 302 Hoechst 33258 179 Hoesch stain procedure 327 Hollow fibre systems 46 Horizontal flow cabinets 172 Hormones 6 HPRT mutants 265 Human blood 98 Hyaluronidase 60

Hybridisation 269 Hybrids 263 Hydroxyurea 235, 259 Hypochlorite 151 Hypoxanthine 230 Idiogram 138 IFN 27, 28 IGF-1, IGF-2 24 Immortality 14 Immunocytochemistry 294 Immunology 5 IMP 265 Impellars 58 In situ hybridisation 271 In vitro rescue of viruses 184 Indicator cells in micoplasma testing 182 Insect cells 93 Insulin-like factors 24, 25 Interferon 9 Interleukins 25, 27, 28 lnterphase 18 Inverted microscope 146 Irradiation 260 Iscove’s medium formulation 324 lsoenzymes 16 lsoleucine starvation 226, 235 ITES 90 Karyotyping 16, 136, 138 KB cells 15 Keratinocytes 226, 303 Kidney epithelial cells 110 Kidney cells primary 108 Kidney tubules 109 Kinetin 94

L cells 14

359

360

CELL CULTURE FOR BIOCHEMISTS

Labelling index 198 suspension cultures 248 LACS cell separator 218 Lactalbumin hydrolysate 79, 94, 314 Lag phase 69 Laminar flow cabinet 171, 172 Laminin 22 Leishman stain 327 Lipofectin 144 Liposomes 144 Liquid emulsion 253 LLC-PKI cells 13 Low density growth 17 Low serum adaptation of cells 90 media 87 Lymphocyte 19, 27, 28, 97 culture 101 fractionation 100 isolation 98 for mab production 271 Lymphokines 27 Lysolecithin 273 Macrophages 112 Mammary epithelial cells 111 Matrix extracellular 19 Maturation promoting factor 195 May-Gnmwald Giemsa 327 McCoy's medium 78 formulation 320 MCDB 402 formulation 319 MCF-7 cells 32 MDCK cells 13 Media 71 CMRL 79 depletion 21 Eagle 75 Ham 78

low serum 91 McCoy 78 MCDB 88 NCTC 79 low serum 91 RPMI 78 stocks 166 Medium 199 formulation 321 Medium CMRL 1066 formulation 323 Medium NCTC 135 formulation 322 MEL cells 301 fractionation 220 MEM 72 Membrane filters 156 Mesenchyme 13 Metabolic cooperation 275 Metasilicate 152 Methocel 20 Methylene blue 234 bacterial stain 327 cellular nucleic acids 334 Metizoate 99 Micoplasma 174 culture 176 effect on cultures 176 elimination 183 tests 177 Microcarrier cultures 65 non-stirred 66 preparation of 65 subculture 67 Microscopy 146 Mitochondria1 preparation 149 Mitogens 14 Mitosis 18, 188 Mitotic index 198 selection 212 Mitsuhashi and Maramorosch 114

SUBJECT INDEX

M ” G 263 Monoclonal antibodies (mab) 5, 271 MOPC 271 Mouse antibody test for viruses 185 Mouse embryo cells 103 MSA 24, 92 Multiplicity of infection (moi) 283 Muscle cells 112, 307 Muslin filter 109 Mutagens 14, 263 Mutants 263 from microcarriers 214 selection of 264 temperature-sensitive 267 MycoTect kit 182 Myelomas 271, 273 Myoblasts 307 NCTC media 79 Neoplastic cells 15 Neuroectodermal cells 13 Neutral red overlay 291 NGF 24 Noble agar 291 Nocodozole 137, 213 Nomarski 147 Non-essential amino acids 76, 117, 314 Nuclear isolation 148 Nutritional requirements 16 Oncogenes 29, 31 One-step growth curve 286 Orcein stain 327 for micoplasmas 178 Osmotic balance 73 Ouabain 271 p34 195 Paracrine response 23 Passage number 16 Patterning of cell monolayers 36 PBS 74, 312

PDGF 23-25 as competence factor 225 Percoll 100 Perfusion systems 58 techniques 38 vessels 45 PHA 101 Phase microscopy 147 Phase-specific mutants 267 Phenol red 313 Phorbol esters 29 Pipette-aid 169 Plant cells 94 Plaque assay 288 Plaque forming units (pfu) 283 Plasma clots 16 Plasmacytomas 271 Plasminogen activator 306 Plastic-coated beads 52 Plating efficiency 117, 118 Pokeweed mitogen 101 Polarity of transport 11 Polyethylene glycol (PEG) 273 Powdered media 77 PPLO agar 177 preparation of 331 Precoated vessels 39 Pressure cooker 155 vessel 158 Primary cells 13, 97 embryo cells 103 Pristane 113 Probabilistic model 193 Pronase 60 Propidium iodide 210 Propiolactone 287 Protein A 293 assay (Bradford) 334 G 293 kinase 29. 30, 195

361

362

CELL CULTURE FOR BIOCHEMISTS

synthesis 249 Pseudorabies virus 283 Pyruvate 117 Q-banding 140 Quiescent cells 18 Quinacrine 140 Radioisotopes 239 Rat embryo cells 103 Rate of DNA synthesis 239 Receptors of growth factors 29 Recovery of cells from liquid nitrogen 134 Repair of DNA 259 Replica plating 268 Replicons 191 Repositories 136 Retinoic acid 306 RFLPs 271 Ribonucleotide reductase 233, 235 RNA assay 333 synthesis 249 Roller apparatus 42 vessels 41 RPMI media 78 Rubber stoppers washing 153 Ruffling movements 20 S-phase 18, 190 blockade 227 Sabouraud fluid medium preparation of 331 Sabouraud’s medium 167 Scintillation vials 39 Scrapers 62 Secreted products 90 Selective killing 215 Semipermeable membranes 39

Sendai virus 273 inactivation 287 production 287 Serum 80 charcoal stripped 84 concentration 21 deprivation 224 dialysed 83, 84 extenders 92 -free medium 84, 85 function of 23 heat inactivation 82 pulse 224 removal of low MW compound 82 SFl medium formulation 324 SGF 24 Shaking for mitotic selection 212 Silicone fluid 65 Siliconised glassware 65 Skin biopsy 102, 304 Soft agar growth of cells in 15 Somatomedins 24, 25 Soy peptone preparation 332 Spira-Cel bottle 42 Spleen cell isolation 272 Split ratio 16 Spreading 19 Stationary cells 236 phase 18 Statistics 3 Stem cells 302 Sterilisation control 155 by filtration 156 hot air 154 tape 154 Sterilising tube Brown 154

SUBJECT INDEX

Sterility checks 166, 331 Steroid hormones 32 Stirring vessels 49 Stripping film 252 Subbed slides 252 Subcellular fractionation 148 Subculture and cell synchronisation 222 Subculturing 13, 59, 63 Substrates 19 Superbeads 51 Suppliers 327 Suspension cells 19 cultures 47 SV40 284 Synchronisation 21 1 by subculture 223 in G2 237 Synchronisation in M 238 Synchrony at G l / S 235 TC plasticware 19 Temperature sensitive mutants 267 Teratocarcinoma cells 305 Terminal cell density 18, 21 TES 75 TGF-u 24 TGF-8 24, 25 Thioglycollate broth 167 Thioguanine 265 Thymidine exhaustion 242 high concentrations 232 kinase mutants 264 phosphorylase 177 uptake 239 Thymid ylate kinase 240 synthetase 231 Tight junctions 11

Tissue extracts 16 TK mutants 264, 266 TNF 27, 28 Top0 inhibition 21 Transfection 142 Transformation 6, 11, 12, 15, 297 and fibronectin 22 Transport of cells 136 of solutes 11 TRICINE 75 TRITC 293 Tritiated thymidine comparison of methods 247 continuous labelling 201 pulse labelling 200 use for DNA synthesis 239 Trypan blue 64, 126 Trypsin 59 Trypsinisation for mitotic selection 213 Trypticase soy broth 167 Tryptose phosphate 71, 314 TSH 29 Tumour cells 18 Ultraviolet irradiation 260 Unbalanced growth 228 Unit gravity separation 217 UV inactivation 287 Vaccines 7 testing and production 296 Versene 61 Vertical flow cabinets 172 Viable cell count 64 Viral contamination 184 transformation 297 Virus classification 280 Virus-infected cells precautions 280

363

364

Virus production 283 Viruses 219 Vital stain 126 Vitronectin 22 Washing glassware 152 W138 cells 14 Winchester bottles 41

CELL CULTURE FOR BIOCHEMISTS

Wounding of cell monolayer 21 Yeast dialysate preparation 332 Yeast extract preparation 332 Zone sedimentation 216