Cell and Tissue Culture: Laboratory Procedures in Biotechnology

~ditedb y The ~ellco~Treus t, London, UK and S~ienti~c ~onsul~aannd cpyu blish in^ Porton S~lisbu~Uy,K Singapore * Toronto Copyright 0 1998 by John Wiley & Sons Ltd, Baffins Lane Chichester, West Sussex P019 1UD, England N707a19ti2 7o 47n37a l International (+441 )2 473 7 9777 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 ~~it~rOijjLaicles 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, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons (Canada) Ltd, 22 Worcester Road, Rexdale, Ontario M9W 1L1, Canada ~ i b roaf ~Co ngress Cataloging-jn-~~blicatioDna ta Cell and tissue culture :l aboratory procedures in biotechnology I edited by Alan Doyle & J. Bryan Griffiths. cpm . . Includes bibliographical references and index. ISBN 0-471-98255-5 (alk. paper) 1. Cellc ulture-Laboratorym anuals. 2. Tissue culture- Laboratory manuals. 1. Doyle, Alan. 11. Griffiths, J. B. ~P248,2S.C44C448 1998 660.6'028-dc21 98-24068 CIP ritis~~ i b r Cu~~ta log~ining ~ ~blicatioDna ta A catalogue record for this book is available from the British Library ISBN 0 471 98255-5 Cover photograph: Electron micrograph courtesy of Mr A.B. Dowsett and Dr T. Battle. CAMR Porton Down, Salisbury Rat hepatocytes in vitro. UK. 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 xiii Foreword xviii Preface XiX Safety xx CHAPTER 1 THE CELL: SELECTION AND STANDARDIZATION 1 1.1 Overview 3 References 4 1.2 Cell Lines for Biotechnologists 5 Introduction 5 Cell line CHO dhfr- 5 Cell line Sf9 6 Cell line Schneider-2 7 Cell lines COS 1/COS 7 7 Cell line NIH3T3 8 Cell line HeLa 8 Cell line J558L 9 Cell line Vero 9 Myeloma cell lines 10 Hybridomas 10 Cell line MRC-5 11 Cell line WI-38 11 Cell line Namalwa 12 Cell line BHK-21 12 Cell line MDCK 13 Cell line GH3 13 Cell line 293 13 Cell line !VCRE/!VCRIP 15 References 15 1.3 Master and Working Cell Banks 18 Scale and composition of cell banks 20 Extended cell bank 20 The cell banking environment and procedures 20 Features required for GLP procedures 21 Conclusion 22 References 24 v1 CELL AND TISSUE CULTURE 1.4 Identity Testing - An Overview 25 Cytogenetic analysis 25 Isoenzyme analysis 26 DNA fingerprinting and DNA profiling 26 References 27 1.5 DNA Fingerprinting 29 PRELIMINARY PROCEDURE: Probe preparation 29 PROCEDURE: Hybridization 30 Discussion 31 References 34 1.6 Detection of Mycoplasma 35 PROCEDURE: DNA stain 36 ALTERNATIVE PROCEDURE: Use of indicator cell lines 39 SUPPLEMENTARY PROCEDURE: Microbiological culture 39 SUPPLEMENTARY PROCEDURE: Elimination of contamination 40 Discussion 40 Ref ereiices 41 1.7 Mycoplasma Detection Methods using PCR 42 PROCEDURE:A mplification 43 SUPPLEMENTARY PROCEDURE: Analysis of amplified samples 44 Discussion 45 References 46 1.8 Bacteria and Fungi 47 PROCEDURE: Detection of bacteria and fungi in cell cultures 47 Discussion 49 References 49 1.9 Elimination of Contamination 50 PROCEDURE: Eradication 50 Discussion 50 References 52 CHAPTER 2 CELL QUANTIFICATION 53 2.1 Overview 55 References 56 2.2 Haemocytometer Cell Counts and Viability Studies 57 PROCEDURE: Haemocytometer cell count 58 Discussion 59 2.3 MTT Assay 62 PROCEDURE: MTT assay - suspension or monolayer cells 62 ALTERNATIVE PROCEDURE: MTT assay - immobilized cells 63 References 64 t L CONTENTS vii 2.4 Neutral Red (NR) Assay 65 PROCEDURE:N eutral red assay 66 SUPPLEMENTARY PROCEDURE:P rotein assay 68 SUPPLEMENTARY PROCEDURE:B ioactivation 68 SUPPLEMENTARY PROCEDURE:U V radiation 69 References 70 2.5 LDH Assay 71 PROCEDURE:M easurement of LDH activity 71 Discussion 73 References 74 2.6 Miniaturized Colorimetric Methods for Determining Cell Number 76 PRELIMINARY PROCEDURE:P retreatment of cells 76 PRELIMINARY PROCEDURE:9 6-Well cell growth or toxicity assays 77 PRELIMINARY PROCEDURET: rypan blue exclusion method for cell viability estimation 77 PROCEDURE:C olorimetric assays: general introduction 78 Discussion 80 References 80 CHAPTER 3 CULTURE ENVIRONMENT 83 3.1 Overview 85 References 86 3.2 Serum-free Systems 87 Elimination of serum 88 Serum substitution 88 Discussion 90 References 91 3.3 Adaptation to Serum-free Culture 92 PRELIMINARY PROCEDURE:M ethod for selecting serum 93 PRELIMINARY PROCEDURE: Method for selecting nutrient medium 94 PRELIMINARY PROCEDURE:T ypes of serum-free media 94 Modifying the nutrient medium 95 PROCEDUREM: ethod for adapting cells to serum-free medium 96 Discussion 97 References 98 3.4 Amino Acid Metabolism 100 PROCEDURE:A mino acid analysis 101 Case study 106 References 107 ... Vlll CELL AND TISSUE CULTURE 3.5 Tissue Culture Surfaces 109 The treatment process 109 St ability 110 Bioactivity 111 Surface choice and comparison 111 Microcarriers 112 Porous membrane systems 113 Discussion 114 References 114 3.6 Plastic and Glass Tissue Culture Surfaces 116 PROCEDUREA: simple procedure for coating surfaces 118 References 120 3.7 Three-dimensional Cell Culture Systems 121 Spheroids 122 Microcarriers 123 Filterwells 124 Matrix sponges or three-dimensional gels and matrix sandwiches 124 Microcontainers 124 Simulated microgravity 125 Conclusion 125 References 125 CHAPTER 4 BIOCHEMISTRY OF CELLS IN CULTURE 129 4.1 Overview 131 4.2 Quantitative Analysis of Cell Growth, Metabolism and Product Formation 133 Errors in calculations 134 Cell growth and death rates 134 Cell metabolism 144 Product formation 156 Concluding remarks 157 Acknowledgements 159 References 159 4.3 Modelling 160 Background for the modelling of mammalian cell cultures 160 Method for kinetic model construction 163 Use of the model for the evaluation of rate-limiting factors 174 Discussion 175 References 178 Background reading 175 4.4 Cell Death in Culture Systems (Kinetics of Cell Death) 179 PROCEDURE: Morphological characterization of cell death 180 PROCEDURE: Biochemical characterization of cell death 182 CONTENTS ix SUPPLEMENTARY PROCEDURE: Purification of apoptotic cells 183 Discussion 184 References 185 4.5 Detoxification of Cell Cultures 187 PROCEDURE: Detoxification by dialysis 187 ALTERNATIVE PROCEDURE: Detoxification by gel filtration 188 Discussion 189 References 189 4.6 Oxygenation 190 PROCEDURE:M easurement of oxygen transfer coefficient and oxygen uptake rate 191 SUPPLEMENTARY PROCEDURE:O xygenation methods 194 References 198 4.7 Mixing 202 Assessing cell damage 202 Parameters used to correlate cell damage due to agitation and/or air sparging 203 Cultures of freely suspended cells 204 Anchorage-dependent cells (microcarrier cultures) 206 References 208 4.8 Mechanical Protection 210 PRELIMINARY PROCEDURE: Additive preparation 21 1 PROCEDURE: Testing before using an additive 21 1 Additives for freely-suspended cells 212 Additives for microcarrier cultures 216 References 216 CHAPTER 5 CULTURE PROCESSES AND SCALE-UP 219 5.1 Overview 221 Scale-up factors 222 Scale-up strategies 222 General principles 224 Monolayer and suspension culture 224 Culture modes 225 Biological factors 225 Summary 226 References 227 5.2 Roller Bottle Culture 228 PROCEDURE: Roller bottle culture of animal cells 228 Comment 229 Supplementary procedures 229 Discussion 230 Background reading 230 X CELL AND TISSUE CULTURE 5.3 Spinner Flask Culture 23 1 PROCEDURE:C ulture of suspension cells in a spinner flask 231 Discussion 234 Background reading 234 5.4 Pilot-scale Suspension Culture of Hybridomas - an Overview 235 Pilot- and large-scale in vitro systems for hybridomas 235 Cultivation modes 237 References 238 5.5 Pilot-scale Suspension Culture of Human Hybridomas 240 PROCEDURE:O ptimization of culture parameters and scaleup 240 Inoculuni preparation and optimization of parameters 241 Discussion 243 References 245 5.6 Chemostat Culture 246 Equipment 248 Method 248 Discussion 251 References 251 5.7 Growth of Human Diploid Fibroblasts for Vaccine Production Multiplate Culture 254 PROCEDURE:P ropagation and subcultivation of human diploid cells in 150-cm’ plastic culture vessels 254 PROCEDURE:S eeding, cultivation, trypsinization and infection of a Nunc 6000-cm2 multiplate unit 255 Discussion 259 References 261 Background reading 261 5.8 Microcarriers - Basic Techniques 262 PRELIMINARY PROCEDURE:S iliconization 264 PROCEDURE:G rowth of cells on microcarriers 265 Discussion 266 References 266 Background reading 267 5.9 Porous Microcarrier and Fixed-bed Cultures 268 PRELIMINARY PROCEDURE:I nitial preparation and calibration of equipment 212 PROCEDURE: Assembly Of culture vessels 272 PROCEDURE:S ystem set-up 274 PROCEDURE:I noculation and maintenance of culture system 274 SUPPLEMENTARY PROCEDURE: Analysis Of consumption and production rates in the fixed-bed porous-glass-sphere culture system 275