Table Of Content~ditedb y
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~ 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
