Animal Cell Culture Methods (eBook)

Front Cover 1
Methods in Cell Biology: Animal Cell Culture Methods 4
Copyright Page 5
Contents 6
Contributors 12
Preface 14
SECTION I: Principles of Cell Culture 16
Chapter 1. Animal Cell Culture Equipment and Techniques 18
I. Introduction 18
II. Equipment 19
III. Laboratory Design 25
IV. Materials 26
V. Cell Culture Methods 28
References 32
Chapter 2. Making Informed Choices: Medium, Serum, and Serum–Free Medium How to Choose the Appropriate Medium and Culture System for the Model You Wish to Create 34
I. Introduction 35
II. The Role of Medium 35
III. pH Control 37
IV. Selecting the Appropriate Medium 37
V. Screening Conditioned Medium for Biological Activity 38
VI. Media Preparation 39
VII. Serum, Plasma, and Other Undefined Additives 40
VIII. Testing Media and Components and Quality Control: "It's in the Water" 40
IX. Troubleshooting Medium Problems 41
X. Altering Commercial Media for Special Uses 42
XI. Medium Optimization 42
XII. Choosing the Optimal Medium: The "Quick and Dirty" Method 43
References 44
Chapter 3. Cell Line Availability: Where to Get the Cell Lines You Need 46
I. Introduction 46
II. Diversity and Selection 47
III. Nomenclature 51
IV. Commercial Utility 52
V. Example Cell Lines Developed 53
VI. Cell Line Cryopreservation and Authentication 55
References 61
Chapter 4. Cell Culture Contamination: Sources, Consequences, Prevention, and Elimination 64
I. Introduction 65
II. Cell Culture Technique 65
III. Cell Culture Contamination 67
IV. Summary 78
References 79
SECTION II: Establishing Cell Lines 82
Chapter 5. Immortalization by Gene Transfection 84
I. Genes Used for Cell Immortalization 85
II. Gene Delivery 92
III. Practical Procedures for Human T-cell Immortalization by Oncogene Transfection 94
IV. Conclusions 98
References 100
Chapter 6. Establishment of Mammalian Testicular Cell Lines 108
I. Introduction 109
II. Establishment of Germ Cell and Testicular Cell Lines Using SV40 Large T Antigen 110
III. In Vitro Seminiferous Cords and Their Use for Promoter Analysis of Germ Cell-Specific Genes 116
IV. Establishment of Germ Cell Lines Using a Combination of the SV40 Large T Antigen and Temperature-Sensitive p53 120
V. Conclusions 122
References 124
Chapter 7. Cell Hybridization, Hybridomas, and Human Hybridomas 126
I. Introduction 127
II. Cell Hybridization 129
III. Hybridomas 133
IV. Human Hybridomas 142
V. Application of Human Monoclonal Antibodies 155
References 157
Chapter 8. Establishing Human Glioma-Derived Cell Lines 162
I. Introduction 162
II. Culture Methodology and General Aspects 163
III. Cell Culture Methodology, Results, and Application 166
IV. Discussion, Review, and Perspectives 174
References 178
Chapter 9. Culture Methods for Selective Growth of Normal Rat and Human Schwann Cells 182
I. Introduction 183
II. Materials and Instruments 185
III. Culture of Rat Schwann Cells from Dorsal Root Ganglia (DRG) 188
IV. Culture of Adult Human Schwann Cells 194
V. Characteristics of Schwann Cell Cultures 196
References 200
Chapter 10. Invertebrate Cell Culture Considerations: Insects, Ticks, Shellfish, and Worms 202
I. Introduction 203
II. Experimental Considerations for the Beginner 205
III. Considerations for All 208
IV. Considerations over the Long Haul 211
References 214
Chapter 11. Cell Line Characterization and Authentication 224
I. Introduction 225
II. Chronology of Cell Line Authentication Efforts 226
III. Markers Used for Cell Line Identification 227
IV. Conclusions 235
References 235
SECTION III: Specialized Culture Techniques 238
Chapter 12. Laboratory Scale-up of Cell Cultures (0.5-50 Liters) 240
I. Introduction 241
II. Roller Bottles 241
III. Microcarriers 242
IV. Growing Cells in Hollow Fibers 244
V. Suspension Adapting Cells 245
VI. Fermentors 247
References 248
Chapter 13. Cell Synchronization 250
I. Introduction 251
II. Estimating Cell Cycle Phase Durations 252
III. Synchronizing Cells 258
IV. Problems in Interpreting Cell Synchrony Experiments 267
References 269
Chapter 14. Measurement of Cell Death 272
I. Introduction 273
II. Cellular Measurement of Apoptosis 274
III. Biochemical Measurement of Apoptosis 280
IV. Concluding Remarks 283
References 284
Chapter 15. Simultaneous Measurement of Cell Cycle and Apoptotic Cell Death 286
I. Introduction 287
II. Methods 289
III. Notes 296
References 297
Chapter 16. Embryonic Stem Cells, Creating Transgenic Animals 300
I. Introduction 301
II. Laboratory Equipment 302
III. Culture Conditions 302
IV. Maintaining Embryonic Stem (ES) Cells 302
V. Preparing Tissue Culture Plates for ES Cells 303
VI. Passaging ES Cells 304
VII. Freezing ES Cells from Confluent 10-cm Tissue Culture Plates 304
VIII. Thawing ES Cells from Freezing Vials 305
IX. Establishing ES Cells 305
X. Introducing DNA into and Selecting for Genetically Altered ES Cells 306
XI. Picking ES Cell Colonies 307
XII. Detecting Genome Alterations 308
XIII. Freezing ES Cells in 96-Well Plates 308
XIV. Thawing ES Cells in 96-Well Plates 309
XV. Chimeras 310
XVI. Preparation of the Aggregation Plate 310
XVII. Preparation of Embryos 311
XVIII. Preparation of ES Cells for Aggregation with Eight-Cell-Stage Mouse Embryos 311
XIX. ES Cell< ->
XX. Germline Transmission 312
XXI. Perspectives 313
References 313
SECTION IV: Microscopy and Morphology 316
Chapter 17. Electron Microscopy: Use of Transmission and Scanning Electron Microscopy to Study Cells in Culture 318
I. Introduction 319
II. Studying Monolayer Cultures with the Transmission Electron Microscope 319
III. Cells Grown on Filters or Matrices 325
IV. Cells Grown in Suspension 326
V. Studying Monolayer Cultures with Scanning Electron Microscopy 329
VI. Cells Grown in Suspension 331
VII. A Few Hints 331
References 332
Chapter 18. Indirect Immunofluorescence Microscopy in Cultured Cells 334
I. Introduction 335
II. Cell Culture 335
III. Sample Preparation for Immunocytochemistry 336
IV. Antibodies 340
V. Signal Detection 342
VI. Miscellaneous Issues 345
VII. Laboratory Protocols for Fixation/Extraction 346
References 352
Chapter 19. Cellular Localization of mRNA and Protein: In Situ Hybridization Histochemistry and in Situ Ligand Binding 354
I. Introduction 355
II. Method: In Situ Hybridization 356
III. Protocol: In Situ Hybridization 361
IV. Method: In Situ Ligand Binding 363
V. Protocol: In Situ Ligand Binding 366
VI. Analysis of Slides 368
VII. Combination of in Situ Hybridization with in Situ Ligand Binding, Immunohistochemistry, or Metabolic Studies 369
References 371
Index 374
Volumes in Series 384