Animal Cell Culture: Essential Methods

Animal Cell Culture......Page 3
Contents......Page 9
Contributors......Page 17
Preface......Page 19
Abbreviations......Page 21
Protocols......Page 27
1.1 Introduction......Page 31
1.2.1 Cell culture laboratory design......Page 33
1.2.1.3 Segregation requirements......Page 34
1.2.1.4 General considerations......Page 35
1.2.2 Services......Page 36
1.2.2.2 Pressurized gases......Page 37
1.2.3.1 Microbiological safety cabinet......Page 38
1.2.3.2 Incubators......Page 44
1.2.3.5 Refrigerators and freezers......Page 46
1.2.3.7 Miscellaneous equipment......Page 47
1.2.4 Culture plasticware and associated small consumable items......Page 48
1.2.4.1 Tissue culture plasticware......Page 49
1.2.4.2 Miscellaneous small items......Page 50
1.2.5 Washing reusable tissue culture equipment......Page 51
1.2.5.1 Pipette washing......Page 52
1.2.6 General care and maintenance of the tissue culture laboratory......Page 53
1.3 Troubleshooting......Page 55
1.3.1 Microbial contamination......Page 56
1.3.1.3 Cleaning up......Page 57
1.3.1.4 Identifying the source......Page 58
1.3.2 Quality control testing......Page 59
References......Page 60
2.1 Introduction......Page 63
2.2.1.1 Autoclaving......Page 64
2.2.2.2 Hot-air ovens......Page 71
2.2.3.2 Gamma rays......Page 73
2.2.4.1 Fumigation......Page 74
2.2.4.2 Liquid disinfectants......Page 76
2.2.5.2 Types of filtration unit......Page 78
2.2.5.3 Filtration set-up......Page 79
2.2.5.4 Testing of bacterial filters......Page 80
2.2.5.5 HEPA filters......Page 81
2.2.6.1 Virus elimination......Page 83
2.2.6.3 Prions......Page 84
2.3 Troubleshooting......Page 86
References......Page 87
3.1.2 Historical context......Page 91
3.2.1 General imaging methodology......Page 92
3.2.1.1 Magnification and resolution......Page 94
3.2.1.2 Illumination......Page 97
3.2.1.3 Contrast......Page 100
3.2.1.4 Aberrations and their corrections......Page 105
3.2.2 Imaging of intact cultured cells in flasks, dishes and plates......Page 107
3.2.3 High-resolution imaging of cells on coverslips......Page 108
3.2.4 Imaging of fluorescently labelled cells......Page 109
3.2.4.2 Microscope configuration for fluorescence......Page 110
3.2.4.3 Three-dimensional imaging......Page 112
3.2.5 Recording of microscopy images......Page 113
3.2.5.1 Time-lapse imaging......Page 114
3.2.5.2 Multidimensional imaging......Page 115
3.2.6.2 Image processing – extraction of morphometric data......Page 116
3.2.7 Maintenance......Page 117
References......Page 118
4.1.1 The terminology of cell and tissue culture......Page 121
4.1.1.1 The origin of cell lines......Page 122
4.1.1.2 The characteristics of normal and transformed cells......Page 123
4.1.2 Basic components of the cell culture environment......Page 125
4.1.2.1 Culture medium......Page 126
4.1.2.3 Stationary versus dynamic media supply......Page 132
4.2.1.1 Working within the microbiological safety cabinet......Page 133
4.2.1.2 Prevention of cellular cross-contamination......Page 137
4.2.2.1 Maintenance of the laboratory......Page 138
4.2.3 Preparation of culture media......Page 142
4.2.4 The culture of attached cells......Page 143
4.2.4.1 Routine culture substrates......Page 144
4.2.4.2 Choice of culture vessels......Page 146
4.2.4.3 Use of attachment factors and biosubstrates......Page 148
4.2.5.1 Adaptation to culture......Page 151
4.2.5.2 Phases of cell growth......Page 152
4.2.6.1 Calculation of in vitro age......Page 153
4.2.6.2 Multiplication rate and population doubling time......Page 155
4.2.6.3 Counting cells in suspension......Page 156
4.2.6.4 Counting/quantifying cells adherent to a substrate......Page 161
4.2.6.5 Expressions of culture ‘efficiency’......Page 164
4.2.7.1 Transporting frozen cells......Page 165
4.2.7.2 Transporting growing cells at ambient temperatures......Page 167
4.2.8.1 Liquid nitrogen......Page 171
4.2.8.2 Pathogens......Page 172
4.3 Troubleshooting......Page 174
References......Page 176
5.1 Introduction......Page 183
5.1.1 Nomenclature definition......Page 184
5.1.3 Impact of culture process and nutrient medium format......Page 186
5.2.1.1 Use of liquid medium and supplemental additives......Page 187
5.2.1.2 Reconstitution of dry-format media......Page 188
5.2.2 Nutrient optimization – determination of basal medium formulation......Page 190
5.2.2.1 Analytical approaches to medium optimization......Page 191
5.2.2.2 Metabolic profiling and statistical analysis as optimization tools......Page 192
5.2.3 Combination of basal medium, nutrient feeds and other process parameters......Page 193
5.2.4 Biological performance screening assays......Page 201
5.2.4.2 Scalability......Page 202
5.2.4.5 Robustness......Page 203
5.3.1.1 Storage......Page 204
5.3.1.3 Sterilization......Page 205
5.3.2 Generality versus specificity – Why cannot a single SFM work for every cell application?......Page 206
5.3.3 Sensitivity to environmental fluctuations – Why are my cells more sensitive when cultured in SFM?......Page 207
5.3.4 Adaptation of cells to SFM - How should I do this, and how and when should banking occur to minimize variability?......Page 208
5.4 Conclusion......Page 210
References......Page 232
6.1 Introduction......Page 215
6.2 Methods and approaches......Page 217
6.2.1 Preparation for cryopreservation......Page 218
6.2.2 Freezing and thawing of cells......Page 223
6.2.4 Safety issues in cell banking......Page 230
6.3 Troubleshooting......Page 231
7.1 Introduction......Page 235
7.2 Methods and approaches......Page 236
7.2.1.1 Adipocytes......Page 237
7.2.1.2 Chondrocytes......Page 239
7.2.1.3 Dermal fibroblasts......Page 241
7.2.1.4 Osteoblasts......Page 243
7.2.1.5 Umbilical vein endothelial cells......Page 246
7.2.2.1 Epithelium......Page 248
7.2.3.1 Hepatocytes......Page 254
7.3 Troubleshooting......Page 255
7.3.3 Culture environment......Page 256
References......Page 259
8.1.1 Uses of cloning......Page 261
8.1.2 Limitations of cloning......Page 262
8.1.3 Special requirements of cells growing at low densities......Page 264
8.2.2 Choice of technique......Page 265
8.2.3.1 Limiting dilution......Page 266
8.2.3.2 Microspot technique......Page 270
8.2.3.3 Cloning by micro-manipulation......Page 271
8.2.4.1 Cloning rings......Page 274
8.2.4.2 Cloning on a hydrophilic FEP surface......Page 276
8.2.5.1 Cloning in soft agar......Page 278
8.2.5.2 Cloning in methylcellulose......Page 280
8.3 Troubleshooting......Page 282
References......Page 283
9.1 Introduction......Page 285
9.2.1.2 Quarantine and receipt of animal cell lines......Page 286
9.2.3 Microbial quality control......Page 289
9.2.3.2 Tests for bacteria, yeasts and fungi......Page 290
9.2.3.3 Tests for mycoplasma......Page 292
9.2.3.4 Virus testing......Page 300
9.2.5 Eradication of contamination......Page 309
9.2.6 Authentication......Page 311
9.2.6.1 Iso-enzyme analysis......Page 313
9.2.6.3 DNA fingerprinting......Page 314
9.2.6.4 Short tandem repeat profiling......Page 315
9.2.7 Regulatory aspects......Page 317
9.3 Troubleshooting......Page 319
9.3.1.2 Provenance and integrity of cell lines......Page 320
9.3.3 Good practice in the cell culture laboratory......Page 321
References......Page 322
10.1 Introduction......Page 327
10.2.1.1 Roller bottles......Page 328
10.2.1.2 Stacked plates......Page 331
10.2.1.3 Microcarriers......Page 333
10.2.2.1 Roller bottles......Page 337
10.2.2.2 Shake flasks......Page 338
10.2.2.3 Spinner flasks......Page 339
10.2.2.4 Bioreactors (fermenters)......Page 340
10.2.2.5 Wave-type bioreactors......Page 344
10.3 Troubleshooting......Page 346
References......Page 348
11.1.1 What if .........Page 353
11.1.2 Chapter aim......Page 354
11.2 Background to GLP......Page 355
11.3.2.1 Management......Page 356
11.3.3 Training......Page 358
11.3.5 Records......Page 359
11.3.6 Test item and test system......Page 360
11.3.8 Archives......Page 361
11.4.1 Planning and initiation......Page 363
11.4.2 Study conduct......Page 365
11.6 Summary......Page 366
References......Page 367
Index......Page 369
Colour plates fall between pages 164 and 165.......Page 379