John wiley sons cell and tissue culture laboratory procedures (NXPowerLite backup)

~ d i t e dby The ~ e l l c o ~ Trust, e London, UK and S ~ i e n t i ~ c ~ o n s u l ~and a n cpublish y in^ Porton S~lisbu~y, UK Singapore * Toronto Copyright 1998 by John Wiley & Sons Ltd, Baffins Lane Chichester, West Sussex P019 1UD, England National 779777 01243 International (+44) 1243 779777 e-mail (for orders and customer services enquiries): cs-books~wiley.co.nk Visit our Home Page on http:/lwww.wiley.co.uk or http://www.wiley.com Reprinted September 1999 All 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, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London, UK WlP 9HE, without the permission in writing of the publisher, The editors and contributors have asserted their right, under the Copyright, Designs and Patents Act 1988, to be identified as the editors of and contributors to this work Other Wiley ~ ~ i t ~ rOjjLices ial John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, USA WILEY-VCH Verlag GmbH, Pappelallee 3, D-69469 Weinheim, Germany Jacaranda Wiley Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons (Canada) Ltd, 22 Worcester Road, Rexdale, Ontario M9W 1L1, Canada ~ i b r oaf ~Congress Cataloging-jn-~~blication Data Cell and tissue culture :laboratory procedures in biotechnology I edited by Alan Doyle & J Bryan Griffiths cm p Includes bibliographical references and index ISBN 0-471-98255-5 (alk paper) Cellculture-Laboratorymanuals Tissue cultureLaboratory manuals Doyle, Alan 11 Griffiths, J B ~P248,2S.C44C448 1998 660.6'028-dc21 98-24068 CIP r i t i s ~~ i b r C~talog~ing u ~ in ~ ~ b l i c a t i oData n A catalogue record for this book is available from the British Library ISBN 471 98255-5 Cover photograph: Electron micrograph courtesy of Mr A.B Dowsett and Dr T Battle CAMR Porton Down, Salisbury UK.Rat hepatocytes in vitro Typeset in 10112pt Times by The Florence Group, Stoodleigh, Devon Printed and bound in Great Britain by Biddles Ltd, Guildford, UK This book is printed on acid-free paper responsibly manufactured from sustainable forestry, in which at least two trees are planted for each one used for paper production Contents Contributors Foreword Preface Safety CHAPTER THE CELL: SELECTION AND STANDARDIZATION 1.1 Overview References 1.2 Cell Lines for Biotechnologists Introduction Cell line CHO dhfrCell line Sf9 Cell line Schneider-2 Cell lines COS 1/COS Cell line NIH3T3 Cell line HeLa Cell line J558L Cell line Vero Myeloma cell lines Hybridomas Cell line MRC-5 Cell line WI-38 Cell line Namalwa Cell line BHK-21 Cell line MDCK Cell line GH3 Cell line 293 Cell line !VCRE/!VCRIP References 1.3 Master and Working Cell Banks Scale and composition of cell banks Extended cell bank The cell banking environment and procedures Features required for GLP procedures Conclusion References xiii xviii XiX xx 5 7 8 9 10 10 11 11 12 12 13 13 13 15 15 18 20 20 20 21 22 24 v1 1.4 1.5 1.6 1.7 1.8 1.9 CELL AND TISSUE CULTURE Identity Testing - An Overview Cytogenetic analysis Isoenzyme analysis DNA fingerprinting and DNA profiling References DNA Fingerprinting PRELIMINARY PROCEDURE: Probe preparation PROCEDURE: Hybridization Discussion References Detection of Mycoplasma PROCEDURE: DNA stain ALTERNATIVE PROCEDURE: Use of indicator cell lines SUPPLEMENTARY PROCEDURE: Microbiological culture SUPPLEMENTARY PROCEDURE: Elimination of contamination Discussion Ref ereiices Mycoplasma Detection Methods using PCR PROCEDURE:Amplification SUPPLEMENTARY PROCEDURE: Analysis of amplified samples Discussion References Bacteria and Fungi PROCEDURE: Detection of bacteria and fungi in cell cultures Discussion References Elimination of Contamination PROCEDURE: Eradication Discussion References CHAPTER CELL QUANTIFICATION Overview 2.1 References Haemocytometer Cell Counts and Viability Studies 2.2 PROCEDURE: Haemocytometer cell count Discussion MTT Assay 2.3 PROCEDURE: MTT assay - suspension or monolayer cells ALTERNATIVE PROCEDURE: MTT assay immobilized cells References 25 25 26 26 27 29 29 30 31 34 35 36 39 39 40 40 41 42 43 44 45 46 47 47 49 49 50 50 50 52 53 55 56 57 58 59 62 62 63 64 t L vii CONTENTS 2.4 2.5 2.6 CHAPTER 3.1 3.2 3.3 3.4 Neutral Red (NR) Assay PROCEDURE:Neutral red assay SUPPLEMENTARY PROCEDURE:Protein assay SUPPLEMENTARY PROCEDURE:Bioactivation SUPPLEMENTARY PROCEDURE:UV radiation References LDH Assay PROCEDURE:Measurement of LDH activity Discussion References Miniaturized Colorimetric Methods for Determining Cell Number PRELIMINARY PROCEDURE:Pretreatment of cells PRELIMINARY PROCEDURE:96-Well cell growth or toxicity assays PRELIMINARY PROCEDURE:Trypan blue exclusion method for cell viability estimation PROCEDURE:Colorimetric assays: general introduction Discussion References 65 66 68 68 69 70 71 71 73 74 CULTURE ENVIRONMENT Overview References Serum-free Systems Elimination of serum Serum substitution Discussion References Adaptation to Serum-free Culture PRELIMINARY PROCEDURE:Method for selecting serum PRELIMINARY PROCEDURE: Method for selecting nutrient medium PRELIMINARY PROCEDURE:Types of serum-free media Modifying the nutrient medium PROCEDURE:Method for adapting cells to serum-free medium Discussion References Amino Acid Metabolism PROCEDURE:Amino acid analysis Case study References 83 85 86 87 88 88 90 91 92 76 76 77 77 78 80 80 93 94 94 95 96 97 98 100 101 106 107 Vlll 3.5 3.6 3.7 CELL AND TISSUE CULTURE Tissue Culture Surfaces The treatment process Stability Bioactivity Surface choice and comparison Microcarriers Porous membrane systems Discussion References Plastic and Glass Tissue Culture Surfaces PROCEDURE:A simple procedure for coating surfaces References Three-dimensional Cell Culture Systems Spheroids Microcarriers Filterwells Matrix sponges or three-dimensional gels and matrix sandwiches Microcontainers Simulated microgravity Conclusion References CHAPTER BIOCHEMISTRY OF CELLS IN CULTURE 4.1 Overview Quantitative Analysis of Cell Growth, Metabolism and 4.2 Product Formation Errors in calculations Cell growth and death rates Cell metabolism Product formation Concluding remarks Acknowledgements References Modelling 4.3 Background for the modelling of mammalian cell cultures Method for kinetic model construction Use of the model for the evaluation of rate-limiting factors Discussion References Background reading Cell Death in Culture Systems (Kinetics of Cell Death) 4.4 PROCEDURE: Morphological characterization of cell death PROCEDURE: Biochemical characterization of cell death 109 109 110 111 111 112 113 114 114 116 118 120 121 122 123 124 124 124 125 125 125 129 131 133 134 134 144 156 157 159 159 160 160 163 174 175 178 175 179 180 182 ix CONTENTS Purification of apoptotic cells Discussion References Detoxification of Cell Cultures PROCEDURE: Detoxification by dialysis ALTERNATIVE PROCEDURE: Detoxification by gel filtration Discussion References Oxygenation PROCEDURE:Measurement of oxygen transfer coefficient and oxygen uptake rate SUPPLEMENTARY PROCEDURE:Oxygenation methods References Mixing Assessing cell damage Parameters used to correlate cell damage due to agitation and/or air sparging Cultures of freely suspended cells Anchorage-dependent cells (microcarrier cultures) References Mechanical Protection PRELIMINARY PROCEDURE: Additive preparation PROCEDURE: Testing before using an additive Additives for freely-suspended cells Additives for microcarrier cultures References 203 204 206 208 210 211 211 212 216 216 CULTURE PROCESSES AND SCALE-UP Overview Scale-up factors Scale-up strategies General principles Monolayer and suspension culture Culture modes Biological factors Summary References Roller Bottle Culture PROCEDURE: Roller bottle culture of animal cells Comment Supplementary procedures Discussion Background reading 219 221 222 222 224 224 225 225 226 227 228 228 229 229 230 230 SUPPLEMENTARY PROCEDURE: 4.5 4.6 4.7 4.8 CHAPTER 5.1 5.2 183 184 185 187 187 188 189 189 190 191 194 198 202 202 X 5.3 5.4 5.5 5.6 5.7 5.8 5.9 CELL AND TISSUE CULTURE Spinner Flask Culture PROCEDURE:Culture of suspension cells in a spinner flask Discussion Background reading Pilot-scale Suspension Culture of Hybridomas an Overview Pilot- and large-scale in vitro systems for hybridomas Cultivation modes References Pilot-scale Suspension Culture of Human Hybridomas PROCEDURE:Optimization of culture parameters and scaleup Inoculuni preparation and optimization of parameters Discussion References Chemostat Culture Equipment Method Discussion References Growth of Human Diploid Fibroblasts for Vaccine Production Multiplate Culture PROCEDURE:Propagation and subcultivation of human diploid cells in 150-cm’ plastic culture vessels PROCEDURE:Seeding, cultivation, trypsinization and infection of a Nunc 6000-cm2 multiplate unit Discussion References Background reading Microcarriers - Basic Techniques PRELIMINARY PROCEDURE:Siliconization PROCEDURE:Growth of cells on microcarriers Discussion References Background reading Porous Microcarrier and Fixed-bed Cultures PRELIMINARY PROCEDURE:Initial preparation and calibration of equipment PROCEDURE: Assembly Of culture vessels PROCEDURE:System set-up PROCEDURE:Inoculation and maintenance of culture system SUPPLEMENTARY PROCEDURE: Analysis O f consumption and production rates in the fixed-bed porous-glass-sphere culture system 231 231 234 234 235 235 237 238 240 240 241 243 245 246 248 248 251 251 254 254 255 259 261 261 262 264 265 266 266 267 268 212 272 274 274 275 318 CELL AND TISSUE CULTURE Envair Ltd York ~ v e n ~ ~ 319 Loughborough Leicestershire LE11 UK Gibco Laboratoires Grand Island Biological Go 3175 Staley Road Grand Island NY 14072 USA Gilson Medical Electronics ( SA Pall Gelman Sciences 72 Rue Gambetta BP 45 F-95400 Villiers le France Northa~ptonNN4 OE Gilson Medical Electronics, Inc 3000 West Beltline USA Pall Gelman Sciences Inc 600 South Wagner Ann Arbor USA ~iddleton W1 53562 USA Grant Instru~ents(Gambridge) Barrington Aire Cool House Spring Gardens London Road on of Life Technolo~ies,Inc UK USA Life Technologies Heraeus E~uipment 111A Corporate South Plain~eld NJ 07080 USA Heraeus Equipment Ltd Unit Brentwood Essex UK 320 CELL AND T I S S ~ E ~ i t a c h Scientific i Instruments Nissei Sangyo America Ltd ountain View CA 94043 USA C~LTUR~ ICN Biomedicals USA ICN/Flow Laboratories PO Box 17 Second Avenue Industrial Estate Irvine KA12 8N U esex TW4 6HJ Imperial Laboratories (Europe) Ltd West ~ o r t w a y Andover Urdorf yClone Laboratories Inc Logan UT 84321 USA Industriezone 111 -9320 Erembodegem-Aalst ~elgium yClone Europe S.A North Nelson Industrial Estate Innovative Chemistry Inc PO Bos 90 ~arshfield A 02050 USA Irvine Scientific 2511 Daimler Street Santa Ana CA 92705 USA IC1 Cellmark ~ i a g n o s t i ~ s ~bingdon O~fordshireOX14 1DY U Jencons (Scientifi~) Ltd ay ~ n ~ u s t r iEstate al Stanbridge Road Leighton Buzzard edfordshire LU7 8UA APPE~DIX 2: ~ O ~ ADDRESSES P ~ ~ Y 321 erck Ltd (E3DH L ~ b o r a t o r y S u ~ ~ l i e s ) inche ester VA 22602 USA UK ~icrobiologicalAssociates Life Sciences Center USA L’Air Liquide 57 av Carnot P13 94503 ~ h a ~ p i g nCedex y France ~ i l l i p o r eCorp USA ~ i l l i ~ o (UK) r e Ltd Allentown PA 18105 ackrnoor Lane USA Life Science Laboratories UK Nalge Company Nalgene Labware E3 fordshire LU4 Life Technolo~ies(Gibco x 68 ~sland NY ~40~~-0068 Andover pshire SPlO 5AA NY 14602-0365 USA Nationa~~ n s t i t ~ t eofs Small Animal Section Veterinary Resources ~uilding14A Room 103 ~ ~ t ~ e MD s d 20205 a USA runswick Scientific Co., Inc edia-Cult Als Symbion Science Park Haraldsgade 68 DK2100 ~ o p e n h a g e ~ Denmark 44 Talmadge Road ox 4005 Edison NJ 08818-4005 USA 32 ne a A 2: c Y 324 CELL AND TISSUE ~ U L T ~ R E hatm man LabSales Techne ( ~ a ~ b r iLtd ~ ~ e ) Collectio~ ~ e r i c a nType Culture Collection ( ~ T C C ) ddress users with same uppliers of cell stocks Culture iken rna~ional el~ct~onic netw major r ~ s o ~cen~res r c ~ are ity, us~allythere are relate This Page Intentionally Left Blank Adaptation to serum-free, 5, 89, 92-98 Adenovirus, 8, 12-13 Adhesion, 116-117 Adventitious agents, 3, 19, 85, 299 Aeration systems, 195 Aeration, membrane, 198, 236 Aeration, surface, 194, 204 Ag~regates,5 Aging, 18 Agitation, 195, 202-204, 208, 210, 236, 244, 285 Airlift culture, 10, 12, 205, 222, 226, 270, 279, 301 Albumin, 87, 95 Amino acid analysis, 101 Amino acid, specific uptake rate, 290 Amino acids, essential, 87, 100, 160 Amino acids, non-essential, 100, 263 Ammonia, 131, 133, 162, 238 Ammonia production, specific rate of, 161, 166, 170, 172 Anchorage dependent cells, 11, 111, 119, 222, 265 Antibiotics, 38, 50-52, 85 Antifoam, 193, 197, 232 Apoptosis, 132, 179, 181-185 Apoptotic bodies, 179-180 ATCC, 3, 9, 49, 296, 325-326 ATP, 56, 151-152, 154-155, 194, 289 Attachment factors, 95 Authentication, 25-27,296 B cells, 10 Bacteria, 19, 47-52 Baculovirus, Batch culture, 133, 142, 163, 171, 225, 237, 242 BHK-21 cells, 12, 14, 192, 262, 302 Binding proteins, 95 Biochemistry, cell, 131, 302 Bioreactor, stirred, 133-134, 164, 203, 224, 236, 241-242, 244, 270, 279, 285, 301 Bovine serum albumin, 216 ubble column reactor, 196, 215 airlift culture see also Carbos~methylcellulose (CMC), 215, 232 CD7,8 Cell bank (see also MC 298 Cell culture suppliers, 326 Cell death kinetics, 179-186 Cell death, 11, 162, 210 (see also Apoptosis and Necrosis) Cell factory (multitray), 119, 226, 230, 254-261 Cell lysis, 55, 140, 175, 180, 202 Cell nuclei count, 55, 264 CellCube, 226, 230 Cell number count, see Haemocytometer Center for Biologics Evaluation &L Research (CBER), 18, 296, 298 Chemostat, 135, 142, 163, 225, 237, 242-244, 246-252, 270, 279 CH0 cells, 5,14, 95, 192, 197, 213, 250, 297, 302 Chromosomes, 25-26, 35, 298 Clonogenic assay, 76 CMRL 1969 medium, 11 Collagen, 124 Co-cultivation, 123 Collagenase, 264 Colorimetric methods,76-81 Company addresses, 315-324, 326 Contact, cell, 131 Contaminants (see Bacteria, Fungi, Mycoplasma, Adventitious agents, Viruses) Contamination, elimination, 50-52 Continuous culture, see Chemostat Coomassie blue stain, 68 COS 1lCOS cells, Coulter counter (see Electronic counting) *Cre/*Crip cells, 15 Cryopreservation, 3, 20 Crystal violet stain, 76, 78-79 330 Culture collections, 1, 325-326 Culture control processes, 282-291 Culture systems, comparison, 226 Cytodex microcarrier,262-263, 265-266 Cytogenetic analysis, 25, 27, 31 Cytoline microcarrier, 268, 270 Cytopathology, 35, 65 Cytotoxicity, 65, 68, 80 Detaxification, 123, 187-189 Dextran, 112, 207, 211, 215-216 Dextranase, 264 Dienes stain, 40 Dih~drofolatereductase, 6, 297 Dilution rate, 136, 139, 246247, 249-250, 275 DMSO, 23, 78, 259 DNA fingerprinting, 19, 23, 29-34, 298 DNA polymerase, 42 DNA stain (see Hoechst) DNA transfection, DON cells, 106 Doubling time, 151, 244 Dry weight, 55 Dulbecco’s modified MEM, 8, 100, 277, 254 Eagle’s basal medium, 11, 12 Eagle’s MEM, 9, 11, 94, 100, 164 EBV, 297 ECACC, 3, 6, 9, 25, 35, 296, 325-326 EDTA, 76 Electronic counting, 56, 77, 78, 93 Endotoxin, 93 Ethanolamine, 95 Exponential growth phase, 187 Extended cell bank, 20 Extracellular matrices, 112, 121 F-12 medium, 5, 90 Fed-batch culture, 133, 225, 237 Fetuin, 89, 95 Fibronectin, 87, 112, 117, 263 Filterwell, 123 Fixed bed culture, 268-281 Flow cytometric analysis, 26 Flow rate, 246 Fluidized bed culture, 268, 278-279 FMDV, 262 Fungi, 19, 47-52 Gene therapy, 13, 302 Genomic DNA, 31-32 GH3 cells, 15 GLP, 21, 296 INDEX Glucose, 131, 146-152, 153, 160-164, 251, 275 Glucose consum~tion,specific rate, 55, 133-4, 161, 170, 172, 275, 289 Glucose-~p~osphate dehydrogenase, 25-26 Glutamine, 131, 133, 160-164, 250 Glutamine, rate of degradation, 161, 163 Glutamine consumption, Specificrate of, 161,166, 170, 172 Glycolysis,151-152 Glycoprotein, 117 Glycosylation, 5, 302 GMP, 21 Good laboratory practice (see GLP) Good manufacturing practice (see GMP) Gram stain, 52 Growth rate, specific, 134-137, 144, 154, 161-163,166,170,172,174,176, 246-247, 249, 289 Growth factors, 87-88, 93, 131 Growth limiting substrate, 246247, 250 Growth yields, 55 Haematoxylin stain, 264 Haemocytometer, 55, 57-61, 77-78, 93, 164 HAT medium, 10 HeLa cells, 8, 25 Henry’s law constant, 151 Hepatocytes, 122-123, 192 HEPES buffer, 85, 263 Heteromyeloma cells, 297 Hoechst stain, 4, 19, 36, 38, 40 Hollow fibre culture, 10, 226, 235 Hormones, 95 HPLC, 105-106, 290 Human diploid cells (HDC), 11, 18, 20, 254-262, 296, 295-298 (see also MRC-5, WI-38) Hybridization, 30, 33, 42, 225 Hybridama cells, 10, 14, 19, 57, 95, 156, 164, 172, 192, 203, 213, 235-245, 250, 270, 289, 297 Identity testing, 25-28, 298 ImmobaSil microcarrier, 268, 270 Immobilized culture, 10, 133, 144, 155-157, 222, 234, 268-281 Immortalization, 10, 297 Impeller, 195, 197, 203-205, 208, 211, 224, 236 Indicator cell lines, 39 Insulin, 89, 94-95, 98 Integrated shear factor, 203 Integrity tests, 21 INDEX Interferon, 6, 14, 221, 259, 266 IS0 9001, 21, 296 Isoenzyme analysis, 19, 25-27, 29, 31, 299 ITS, 95 J558L cells, 9, 14 Karyology, 19, 26, 298-299 aryotype, 25, Kinetic model, 160-178 &,a, 134, 191, 193, 195 Kolmogorov eddy, 203-204, 207-208 Lactate, 71, 148, 150, 152-153, 162-164, 275 Lactate de~ydrogenase (LDH),55, 140-142, 202 Lactate production, specific rate of, 161, 166, 170, 172 L 202 71-74, 175, L L L Liquid flow control, Liquid level control, Lysosomes, 65-66 331 Mixing, 202-209, 210, 222, 231 ~odelling,see Kinetic model Moloney murine leukaemia virus, 15 Monoclonal antibodies, 10, 14, 187, 237, 325 MRC-5 cells, 11, 14, 111, 254, 265 MTT, 56, 62-44, 76, 79, 80 ~ultilocusDNA, 26, 34, 298 Mycoplasma, 3-4, 19, 35-46, 50-52, 299 Myeloma cells, 9, 89, 296 Namalwa cells, 12, 14 Necrosis, 132, 179, 181-182 Neutral red, 56, 65-70,'76,78-79 NIH/3T3 cells, 8, 14 NSO cells, 10, 14 Nuclear magnetic resonanc~,153 Nutrient uptake, specific rate, 162 Oligonucleotide probes, 32 Osmotic pressure, 162, 288 Oxygen, 12, 131, 151, 153, 190-199, 222, 226, 243, 286 O~ygenation,190-199, 205, 236 xygen consumption rate, 55, 151, 190-192 Oxygen, dissolved 242, 284-285 Oxygen tension, Oxygen transfer, 194, 198, olyvinylpyrro~don~s, 213 Mitotic index, 55 359 332 Porous membrane culture, 113-114 Porous microcarrier, 119, 123, 206, 226, 268-281 Process control, 282-286 Productivity, 278-279, 301-303 Quality control, 3, 18-19, 23, 29, 31, 40, 295 Rabies virus, 12 adiochromium, 56 APD, 298 Rate limiting factors, 174 Recombinant cell lines, Recombinant proteins, 187, 221 Redox, 56 R e ~ u l a t guidelines, o~ 29 esidual DNA, 299 eynold’s number, 204 obotics, 230 oiler bottle culture, 10, 118-119, 222, 228-230, 262 otating wall vessel, 12 oux bottle, 116, 164 PM1 1640 medium, 10, 12, 94, 100, 164 Sarcoma virus, Scale-up, 119, 221-227, 242, 278 Schneider-2 cells, Seed lot system, 18, 22 Seleniu~, 94-95, 98 Semi-continuous culture, 139 Sendai virus, 12 Sensors, 282 erum, 85, 87, 213 Serum factors, 117 Serum~freemedium, 7, 87-99 Serum, substitution, 88-90, 94-95 Sf9 cells, Siliconi~ation,233, 263-264 Siran glass spheres, 270-279 SOP, 22, 23 Soybean trypsin inhibitor, 93, 266 S~arging,i f ,194, 196197, 203, 205-20~, 222, 236, 244 Specific death rate, 134, 162, 166, 170, 172, 175-176, 247 Specific product formation rate, 145, 156, 275 Specific rate of antibody production, 161, 166, 170, 172, 289 INDEX Specific utilization rate, 55 Spheroids, 121-123, 144 Spin filter, 223, 226, 238, 266 Spinner culture, 7, 205, 222, 231-234 Spinner flasks, 118, 208, 231-234, 241, 263, 265 Standard operating procedure (see SOP) Steady-state, 140, 238, 247, 250-251 Sterility tests, 19 Stirrer speed, 234, 243-244, 265, 285 Stoichometric coefficient, 172 Subculture, 23 Sulphorhodamine B stain, 76, 78-79 Suppliers, see Company Addresses urfaces, tissue culture, 109-120 SV40, 8, 295 Temperature control, 283 Terminology, 305-313 TGF-P, 89 Trace elements, 95 Transfected cell lines, Transferrin, 94-95, Transformatio~,13, 35 Trypan blue, 55, 57-61, 77-78, 80, 179, 202 Trypsin, 77 Tryptose phosphate broth, 12, 263 UV radiation, 69 Verax system, 268-270 Vero cells, 9, 39, 134, 192, 295 Viability, 19, 55-62, 65, 67, 76, 136, 140, 210, 286 Viral conta~nants,92, 297 Viral vaccines, 9, 11, 14, 221, 254-262, 266, 295 Viruses, 49 Vital stains, 55 VNTR’s, 27 WCB, 18, 20, 22, 245, 254-255, 259, 298 CC, 326 WHO, 18,295-296,298-299 WI-38 cells, 11, 14, 254, 295 Working cell bank (see WCB) Yield, cell, 154, 247 Yield, product, 157 ... NIH3T3 Cell line HeLa Cell line J558L Cell line Vero Myeloma cell lines Hybridomas Cell line MRC-5 Cell line WI-38 Cell line Namalwa Cell line BHK-21 Cell line MDCK Cell line GH3 Cell line 293 Cell. .. th applied cell culture make it a useful a~junct to traini~g programmes inthe cell culture laboratory Thecomprehensivemanual ? ?Cell and Tissue Culture: LaboratoryProcedures’ Griffiths and D.G Newell,... contamination by other animal cells or microorganis~s, theoriginal pure culture will not be retrievable Cell and Tissue Culture: Laboratory Procedures 1998 John Wiley & Sons Ltd in ~ i o t e c ~