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Culture of animal cells : a manual of basic technique and specialized applications / R. Ian Freshney. — 6th ed. — Hoboken, N.J. : Wiley-Blackwell ; Chichester : John Wiley [distributor], 2010. – (59.105/F885/6th ed.) |
Contents
Contents
List of Figures， xix
List of Color Plates， xxiii
List of Protocols， xxv
Preface and Acknowledgements， xxvii
Abbreviations， xxix
1. Introduction， 1
1.1. Historical Background， 1
1.2. Advantages of Tissue Culture， 6
1.3. Limitations， 7
1.4. Major Differences In vitro， 8
1.5. Types of Tissue Culture， 8
2. Biology of Cultured Cells， 11
2.1. The Culture Environment， 11
2.2. Cell Adhesion， 11
2.3. Cell Proliferation， 15
2.4. Differentiation， 16
2.5. Cell Signaling， 17
2.6. Energy Metabolism， 19
2.7. Origin of Cultured Cells， 20
3. Laboratory Design， Layout， and Equipment， 25
3.1. Layout， Furnishing， and Services， 25
3.2. Layout， 30
4. Equipment and Materials， 37
4.1. Requirements of a Tissue Culture Laboratory， 37
4.2. Aseptic Area， 37
4.3. Incubation and Culture， 47
4.4. Preparation and Sterilization， 50
4.5. Storage， 53
4.6. Supplementary Laboratory Equipment， 55
4.7. Specialized Equipment， 56
5. Aseptic Technique， 57
5.1. Objectives of Aseptic Technique， 57
5.2. Elements of Aseptic Environment， 58
5.3. Sterile Handling， 61
5.4. Standard Procedure， 65
5.5. Apparatus and Equipment， 69
6. Safety， Bioethics， and Validation， 71
6.6. Fire， 78
6.7. Ionizing Radiation， 78
6.8. Biohazards， 79
6.9. Bioethics， 86
6.10. Quality Assurance， 87
6.11. Validation， 87
7. Culture Vessels and Substrates， 89
7.1. The Substrate， 89
7.2. Treated Surfaces， 90
7.3. Choice of Culture Vessel， 91
7.4. Specialized Systems， 96
8. Defined Media and Supplements， 99
8.1. Development of Media， 99
8.2. Physicochemical Properties， 99
8.3. Balanced Salt Solutions， 107
8.4. Complete Media， 107
8.5 Serum， 109
8.6 Selection of Medium and Serum， 111
8.7 Other Supplements， 114
9. Serum-Free Media 115
9.1. Disadvantages of Serum， 115
9.2. Advantages of Serum-Free Media， 121
9.3. Disadvantages of Serum-Free Media， 122
9.4. Replacement of Serum， 122
9.5. Selection of Serum-Free Medium， 124
9.6. Development of Serum-Free Medium， 124
9.7. Preparation of Serum-Free Medium， 129
9.8. Animal Protein-Free Media， 129
9.9. Conclusions， 132
10. Preparation and Sterilization 133
10.1. Preparation of Reagents and Materials， 133
10.2. Sterilization of Apparatus and Liquids， 133
10.3. Apparatus， 134
10.4. Reagents and Media， 140
10.5. Sterilization of Media， 151
10.6. Control， Testing， and Storage of Media， 160
11. Primary Culture， 163
11.1. Initiation of a Primary Cell Culture， 163
11.2. Isolation of the Tissue， 164
11.3. Types of Primary Culture， 170
12. Subculture and Cell Lines， 187
12.1. Subculture and Propagation， 187
12.2. Choosing a Cell line， 193
12.3. Routine Maintenance， 193
12.4. Subculture， 196
13. Cloning and Selection， 207
13.1. Cell Cloning， 207
13.2. Stimulation of Plating Efficiency， 209
13.3. Suspension Cloning， 214
13.4. Isolation of Clones， 218
13.5. Replica Plating， 221
13.6. Selective Inhibitors， 221
13.7. Isolation of Genetic Variants， 223
13.8. Interaction with Substrate， 224
14. Cell Separation， 227
14.1. Cell Density and Isopyknic Sedimentation， 227
14.2. Cell Size and Sedimentation Velocity， 230
14.3. Antibody-Based Techniques， 232
14.4. Fluorescence-Activated Cell Sorting， 234
14.5. Other Techniques， 236
14.6. Beginner's Approach to Cell
Separation， 237
15. Characterization， 239
15.1. The Need for Characterization， 239
15.2. Authentication， 239
15.3. Record Keeping and Provenance， 240
15.4. Parameters of Characterization， 240
15.5. Cell Morphology， 242
15.6. Confocal Microscopy， 253
15.7. Chromosome Content， 253
15.8. DNA Analysis， 256
15.9. RNA and Protein Expression， 261
15.10. Enzyme Activity， 261
15.11. Antigenic Markers， 267
15.12. Differentiation， 268
16. Differentiation， 269
16.1. Expression of the In vivo Phenotype， 269
16.2. Stages of Differentiation， 270
16.3. Proliferation and Differentiation， 270
16.4. Commitment and Lineage， 270
16.5. Stem Cell Plasticity， 271
16.6. Markers of Differentiation， 272
16.7. Induction of Differentiation， 272
16.8. Differentiation and Malignancy， 278
16.9. Practical Aspects， 278
17. Transformation and Immortalization， 279
17.1. Role in Cell Line Characterization， 279
17.2. What is Transformation?， 279
17.3. Genetic Instability and Heterogeneity， 279
17.4. Immortalization， 281
17.5. Aberrant Growth Control， 290
17.6. Tumorigenicity， 293
18. Contamination， 299
18.1. Sources of Contamination， 299
18.2. Types of Microbial Contamination， 301
18.3. Monitoring for Contamination， 301
18.4. Disposal of Contaminated Cultures， 311
18.5. Eradication of Contamination， 311
18.6. Cross-contamination， 315
18.7. Conclusions， 315
19. Cryopreservation， 317
19.1. Rationale for Freezing， 317
19.2. Considerations before
19.3. Principles of Cryopreservation， 318
19.4. Vitrification， 327
19.5. Design and Control of Freezer Stocks， 331
19.6. Cell Banks， 332
19.7. Transporting Cells， 333
20. Quantitation， 335
20.1. Cell Counting， 335
20.2. Cell Weight， 344
20.3. DNA Content， 344
20.4. Protein， 345
20.5. Rates of Synthesis， 346
20.6. Preparation of Samples for Enzyme Assay and Immunoassay， 348
20.7. Cytometry， 348
20.8. Replicate Sampling， 348
20.9. Cell Proliferation， 349
20.10. Plating Efficiency， 357
20.11. Labeling Index， 360
20.12. Cell Cycle Time， 363
20.13. Cell Migration， 363
21. Cytotoxicity， 365
21.1. Viability， Toxicity， and Survival， 365
21.2. In vitro Limitations， 366
21.3. Nature of the Assay， 366
21.4. Applications of Cytotoxicity Assays， 377
21.5. Genotoxicity， 377
21.6. Inflammation， 380
22. Specialized Cells， 383
22.1. Cell Culture of Specialized Cells， 385
22.2. Epithelial Cells， 385
22.3. Mesenchymal Cells， 408
22.4. Neuroectodermal Cells， 422
22.5. Hematopoietic Cells， 430
22.6. Gonads， 432
23. Stem Cells， Germ Cells， and Amniocytes， 433
23.1. Stem Cells， 433
23.2. Germ Ceils， 445
23.3. Extraembryonic Cells， 445
24. Culture of Tumor Cells， 463
24.1. Problems of Tumor Cell Culture， 463
24.2. Sampling， 464
24.3. Disaggregation， 465
24.4. Primary Culture， 465
24.5. Selective Culture of Tumor Cells， 466
24.6. Development of Cell Lines， 468
24.7. Characterization of Tumor Cell
24.8. Specific Tumor Types， 471
25. Three-Dimensional Culture， 481
25.1. Cell Interaction and Phenotypic Expression， 481
25.2. Organ Culture， 482
25.3. Histotypic Culture， 486
25.4. Organotypic Culture， 493
25.5. Imaging Cells in 3-D Constructs， 495
26. Scale-up and Automation， 497
26.1. Scale-up in Suspension， 497
26.2. Scale-up in Monolayer， 503
26.3. Process Control， 510
26.4. Automation， 513
27. Specialized Techniques， 517
27.1. Lymphocyte Preparation， 517
27.2. Autoradiography， 518
27.3. Time-Lapse Recording， 522
27.4. Cell Synchrony， 525
27.5. Culture of Cells from Poikilotherms， 525
27.6. Somatic Cell Fusion， 527
27.7. Production of Monodonal Antibodies， 529
28. Training Programs， 533
28.1. Objectives， 533
28.2. Preparative and Manipulative Skills， 533
28.3. Basic Cell Culture Techniques， 543
28.4. Advanced Exercises， 557
28.5. Specialized Exercises， 568
29. Problem Solving， 569
29.1. Abnormal Appearance of Cells， 570
29.2. Slow Cell Growth， 570
29.3. Medium， 572
29.4. Substrates and Containers， 575
29.5. Microbial Contamination， 576
29.6. Chemical Contamination， 581
29.7. Primary Culture， 582
29.8. Differentiation， 583
29.9. Feeding， 584
29.10. Subculture， 585
29.11. Cloning， 586
29.12. Cross-contamination and Misidentification， 588
29.13. Cryopreservation， 588
29.14. Cell Counting， 590
30. In Conclusion， 593
Appendix I: Calculations and Preparation of Reagents， 595
Appendix II: Sources of Equipment and Materials， 603
Appendix III: Suppliers and Other Resources， 623
Appendix IV: Glossary， 633
Appendix V: Cross-contaminated or Misidentified Cell Lines， 639
Appendix VI: General Textbooks and Relevant Journals， 661
References 663
Index 717
