Culture of animal cells: a manual of basic technique and specialized applications
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| Format: | Buch |
| Sprache: | English |
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Wiley Blackwell
[2016]
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| Ausgabe: | Seventh edition |
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | xxxviii, 684 Seiten Illustrationen, Diagramme |
| ISBN: | 9781118873656 |
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Datensatz im Suchindex
| _version_ | 1804174917865832448 |
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| adam_text | Titel: Culture of animal cells
Autor: Freshney, Robert Ian
Jahr: 2016
Contents
List of Figures, xix
List of Color Plates, xxiii
List ofTables, xxv
List of Protocols, xxvii
List of Minireviews, xxix
Preface and Acknlowledgments, xxxi
Abbreviations, xxxiii
About the Companion Website xxxviii
1. Introduction, 1
1.1 Historical Background, 2
1.2 Advantages of Tissue Culture, 8
1.2.1 Control of the Environment, 8
1.2.2 Characterization and Homogeneity
of Sample, 8
1.2.3 Economy, Scale, and Mechanization, 9
1.2.4 In vitro Modeling of In vivo Conditions, 9
1.3 Limitations, 9
1.3.1 Expertise, 9
1.3.2 Quantity, 9
1.3.3 Dedifferentiation and Selection, 10
1.3.4 Origin of Cells, 10
1.3.5 Instability, 10
1.4 Major Differences In Vitro, 10
1.5 Types of Tissue Culture, 11
References, 13
2. Biologv of Cultured Cells, 19
2.1 The Culture Environment, 19
2.2 Cell Adhesion, 19
2.2.1 Cell Adhesion Molecules, 19
2.2.2 Intercellular Junctions, 21
2.2.3 Cytoskeleton, 21
2.2.4 Extracellular Matrix, 22
2.2.5 Cell Motility, 23
2.3 Cell Proliferation, 23
2.3.1 Cell Cycle, 23
2.3.2 Control of Cell Proliferation, 24
2.4 Differentiation, 24
2.4.1 Induction and Maintenance of
Differentiation, 26
2.4.2 Plasticity of Differentiation and
Dedifferentiation, 27
2.5 Cell Signaling, 28
2.6 Energy Metabolism, 28
2.7 Origin of Cultured Cells, 30
2.7.1 Initiation of the Culture, 30
2.7.2 Evolution of Cell Lines, 31
2.7.3 Senescence, 32
2.7.4 Transformation and the Development of
Continuous Cell Lines, 32
2.8 Definitions, 33
References, 34
Supplier, 36
3. Laboratory Design and Layout 37
3.1 Planning, Furnishing, and Services, 37
3.1.1 Requirements, 38
3.1.2 Services, 41
3.1.3 Ventilation, 42
vii
viii
CONTENTS
3.2 Design and Layout, 43
3.2.1 Sterile Handling Area, 43
3.2.2 Laminar Flow Hoods, 43
3.2.3 Service Bench, 44
3.2.4 Quarantine and Containment, 44
3.2.5 Incubation, 44
3.2.6 Preparation Area, 46
3.2.7 Storage, 47
3.3 Disaster Management, 49
References, 49
4. Equipment and Materials, 51
4.1 Requirements of a Tissue Culture
Laboratory, 51
4.2 Sterile Working Area, 51
4.2.1 Laminar-Flow BSC, 51
4.2.2 Service Carts, 55
4.2.3 Sterile Liquid Handling—Pipetting and
Dispensing, 56
4.2.4 Inverted Microscope, 59
4.2.5 Camera and Monitor, 59
4.2.6 Dissecting Microscope, 60
4.2.7 Centrifuge, 60
4.2.8 Cell Counting, 62
4.3 Incubation and Culture, 63
4.3.1 Incubator, 63
4.3.2 Humid COz Incubator, 63
4.3.3 Temperature Recorders, 65
4.3.4 Roller Racks, 65
4.3.5 Magnetic Stirrer, 66
4.3.6 CultureVessels, 66
4.4 Preparation and Sterilization, 66
4.4.1 Washup, 66
4.4.2 Preparation of Media and Reagents, 67
4.4.3 Sterilization, 69
4.5 Storage, 70
4.5.1 Consumables, 70
4.5.2 Refrigerators and Freezers, 70
4.5.3 Cryostorage Containers, 70
4.5.4 Controlled-Rate Freezer, 71
4.6 Supplementary Laboratory Equipment, 71
4.6.1 Computers and Networks, 71
4.6.2 Upright Microscope, 71
4.6.3 Low-Temperature Freezer, 71
4.6.4 Confocal Microscope, 71
4.6.5 PCR Thermal Cycler, 72
4.7 Specialized Equipment, 72
4.7.1 Microinjection Facilities, 72
4.7.2 Colony Counter, 72
4.7.3 Centrifugal Elutriator, 72
4.7.4 Flow Cytometer, 72
References, 72
Suppliers, 72
5. Aseptic Technique, 73
5.1 Objectives of Aseptic Technique, 73
5.1.1 Risk of Contamination, 73
5.1.2 Maintaining Sterility, 74
5.2 Elements of Aseptic Environment, 75
5.2.1 Laminar Flow, 75
5.2.2 QuietArea, 77
5.2.3 Work Surface, 77
5.2.4 Personal Hygiene, 77
5.2.5 Reagents and Media, 78
5.2.6 Cultures, 79
5.3 Sterile Handling, 79
5.3.1 Swabbing, 79
5.3.2 Capping, 79
5.3.3 Fläming, 79
5.3.4 Handling Botties and Flasks, 80
5.3.5 Pipetting, 80
5.3.6 Large-Volume Dispensing, 81
5.3.7 Pouring, 81
5.4 Standard Procedure, 81
Protocol 5.1. Aseptic Technique In BSC, 82
Protocol 5.2. Working On The Open Bench, 84
5.4.1 Culture Flasks and Botties, 85
5.4.2 Petri Dishes and Multiwell Plates, 85
Protocol 5.3. Handling Dishes or Plates, 86
5.5 Apparatus and Equipment, 86
5.5.1 Refrigerators and Coldrooms, 86
5.5.2 Incubators, 86
Protocol 5.4. Cleaning Incubators, 87
5.5.3 Boxed Cultures, 87
5.5.4 Gassing with C02, 87
References, 88
Suppliers, 88
6. Safety, Biaethics, and Validation, 89
6.1 Laboratory Safety, 89
6.2 Risk Assessment, 89
6.3 Standard Operating Procedures, 91
6.4 Safety Regulations, 91
6.5 General Safety, 92
6.5.1 Operator, 93
6.5.2 Equipment, 93
6.5.3 Glassware and Sharp Items, 94
6.5.4 Chemical Toxicity, 94
6.5.5 Gases, 95
6.5.6 Liquid Nitrogen, 95
6.5.7 Burns, 96
6.6 Fire, 96
6.7 lonizing Radiation, 97
6.7.1 Ingestion, 97
6.7.2 Disposal of Radioactive Waste, 97
6.7.3 Irradiation from Labeled Reagents, 97
6.7.4 Irradiation from High-Energy Sources, 97
CONTENTS iX
6.8 Biohazards, 97
6.8.1 Levels of Biological Containment, 98
6.8.2 Biological Safety Cabinets (BSCs), 98
6.8.3 Human Biopsy Material, 101
6.8.4 Cell Lines, 104
6.8.6 Genetic Manipulation, 104
6.8.6 Disposal of Biohazardous Waste, 104
6.8.7 Decontamination and Fumigation, 104
6.9 Bioethics, 104
6.9.1 Animal Tissue, 105
6.9.2 Human Tissue, 105
6.10 Quality Assurance, 106
6.10.1 Procedures, 107
6.10.2 Quality Control (QC), 107
6.10.3 Validation, 107
6.10.4 Authentication, 107
6.10.5 Provenance, 108
6.10.6 Contamination, 108
References, 108
Suppliers, 110
7. Culture Vessels and Substrates, III
7.1 The Substrate, 111
7.1.1 Attachment and Growth, 111
7.1.2 Common Substrate Materials, 111
7.1.3 Alternative Substrates, 112
7.2 Treated Surfaces, 112
7.2.1 Substrate Coating, 112
Protocol 7.1. Preparation of ECM, 113
7.2.2 Feeder Layers, 114
7.2.3 Nonadhesive Substrates, 115
7.3 Choice of Culture Vessel, 115
7.3.1 CellYield, 116
7.3.2 Multiwell Plates, 118
7.3.3 Flasks and Petri Dishes, 118
7.3.4 HighYields, 118
7.3.5 Suspension Culture, 118
7.3.6 Venting, 119
7.3.6 Sampling and Analysis, 120
7.3.7 Uneven Growth, 120
7.3.8 Cost, 120
7.4 Specialized Systems, 121
7.4.1 Permeable Supports, 121
7.4.2 Three-Dimensional Matrices, 121
References, 122
Suppliers, 124
8. Defined Media and Supplements, 125
8.1 Development of Media, 125
8.2 Physicochemical Properties, 125
8.2.1 pH, 125
Protocol 8.1. Preparation of pH Standards, 126
8.2.2 CO, and Bicarbonate, 126
8.2.3 Buffering, 127
8.2.4 Oxygen, 128
Minireview 8.1. Hypoxie Cell Culture, 128
8.2.5 Osmolality, 131
8.2.6 Temperature, 132
8.2.7 Viscosity, 132
8.2.8 Surface Tension and Foaming, 133
8.3 Balanced Salt Solutions, 133
8.4 Complete Media, 134
8.4.1 AminoAcids, 134
8.4.2 Vitamins, 134
8.4.3 Salts, 134
8.4.4 Glucose, 139
8.4.5 Organic Supplements, 139
8.4.6 Hormones and Growth Factors, 139
8.4.7 Antibiotics, 139
8.5 Serum, 140
8.5.1 Protein, 140
8.5.2 Growth Factors, 142
8.5.3 Hormones, 142
8.5.4 Nutrients and Metabolites, 142
8.5.5 Lipids, 142
8.5.6 Minerals, 142
8.5.7 Inhibitors, 142
8.6 Selection of Medium and Serum, 142
8.6.1 Serum Batch Reservation, 143
8.6.2 Testing Serum, 144
8.6.3 Heat Inactivation, 145
8.7 Other Supplements, 145
8.7.1 Amino Acid Hydrolysates, 145
8.7.2 Embryo Extract, 145
8.7.3 Conditioned Medium, 145
8.8 Storage, 145
References, 145
Suppliers, 148
9. Serum-Free Media, 149
9.1 Disadvantages of Serum, 149
9.2 Advantages of Serum-Free Media, 154
9.3 Disadvantages of Serum-Free Media, 155
9.4 Replacement of Serum, 155
9.4.1 Commercially Available Serum-Free
Media, 155
9.4.2 Serum Replacements, 155
9.4.3 Serum-Free Subculture, 157
9.4.4 Hormones, 157
9.4.5 Growth Factors, 157
9.4.6 Nutrients in Serum, 161
9.4.7 Proteins and Polyamines, 161
9.4.8 Viscosity, 161
X
CONTENTS
9.5 Development of Serum-Free Medium, 162
9.6 Selection of Serum-Free Medium, 162
9.6.1 Cell or Product Specificity, 162
9.6.2 Adaptation of Cell Lines to
Serum-Free Media, 162
9.7 Preparation of Serum-Free Medium, 165
9.8 Animal Protein-Free Media, 165
9.9 Conclusions, 165
References, 166
Suppliers, 170
10. Preparation and Sterilization, 173
10.1 Preparation of Reagents and Materials, 173
10.2 Sterilization ofApparatus and Liquids, 173
10.2.1 Dry-Heat Sterilization, 174
10.2.2 Pressurized-Steam Sterilization, 174
10.2.3 Irradiation, 175
10.2.4 Chemical Sterilization, 175
10.2.5 Sterility Indicators, 175
10.2.6 Filter Sterilization, 176
10.3 Apparatus, 176
10.3.1 Glassware, 176
Protocol 10.1. Preparation and Sterilization of
Glassware, 178
10.3.2 Glass Pipettes, 179
Protocol 10.2. Preparation and Sterilization of
Glass Pipettes, 179
10.3.3 Screw Caps, 180
Protocol 10.3. Preparation and Sterilization of
Screw Caps, 181
10.3.4 Selection of Detergent, 182
10.3.5 Miscellaneous Equipment, 183
10.3.6 Reusable Sterilizing Filters, 185
Protocol 10.4. Sterilizing Filter Assemblies, 185
10.4 Reagents and Media, 186
10.4.1 Water, 186
Protocol 10.5. Preparation and Sterilization of
Ultrapure Water (UPW), 187
10.4.2 Maintenance ofWater Purifier, 187
10.4.3 Balanced Salt Solutions, 188
Protocol 10.6. Preparation and Sterilization of
D-PBSA, 188
10.4.4 Preparation and Sterilization of
Media, 189
Protocol 10.7. Preparation of Medium from 1x
Stock, 189
Protocol 10.8. Preparation of Medium from 10x
Concentrate, 190
Protocol 10.9. Preparation of Medium from
Powder, 192
10.5 Sterilization of Media, 192
10.5.1 Autoclavable Media, 192
10.5.2 Sterile Filtration, 192
Protocol 10.10. Sterile Filtration with Syringe-Tip
Filter, 195
Protocol 10.11. Sterile Filtration with Vacuum Filter
Flask, 196
Protocol 10.12. Sterile Filtration with Small Inline
Filter, 196
Protocol 10.13. Sterile Filtration with Large Inline
Filter, 198
10.5.3 Serum, 199
10.5.4 Preparation and Sterilization of Other
Reagents, 199
10.6 Control, Testing, and Storage of Media, 199
10.6.1 Quality Control, 199
10.6.2 Sterility Testing, 199
10.6.3 Culture Testing of Medium and
Serum, 200
Protocol 10.14. Testing Medium hy Plating
Efficiency, 201
Protocol 10.15. Testing Medium by Growth, 202
10.6.4 Storage, 204
References, 204
Suppliers, 205
SupptementarY Material*
Preparation and Sterilization of Serum
Protocol 10.16-Su. Collection and Sterilization
of Serum
Dialysis of Serum
Protocol 10.17-Su. Dialysis of Serum
Suppliers
11. Printary Culture, 207
11.1 Initiation of a Primary Cell Culture, 207
11.1.1 Proteases Used in Disaggregation, 207
11.1.2 Common Features of Disaggregation, 209
11.2 Isolation of the Tissue, 209
11.2.1 Mouse Embryo, 210
Protocol 11.1. Isolation of Mouse Embryos, 211
11.2.2 Chick Embryo, 214
Protocol 11.2. Isolation of Chick Embryos, 214
11.2.3 Fluman Biopsy Material, 214
Protocol 11.3. Handling Human Biopsies, 215
11.3 Types of Primary Culture, 216
11.3.1 Primary Explantation, 216
Protocol 11.4. Primary Explants, 216
11.3.2 Enzymatic Disaggregation, 219
11.3.3 WarmTrypsin, 219
Protocol 11.6. Tissue Disaggregation in Warm
Trypsin, 219
11.3.4 Trypsinization with Cold
Preexposure, 221
CONTENTS *# Xi
Protocol 11.7. Tissue Disaggregation in Cold
Trypsin, 222
11.3.5 Chick Embryo Organ Rudiments, 223
Protocol 11.8. Chick Embryo Organ
Rudiments, 224
11.3.6 Other Enzymatic Procedures, 227
11.3.7 Collagenase, 227
Protocol 11.9. Tissue Disaggregation in
Collagenase, 227
11.3.8 Mechanical Disaggregation, 229
Protocol 11.10. Mechanical Disaggregation By
Sieving, 229
Protocol 11.11. Enrichment of Viable Cells, 230
11.3.9 Separation of Viable and Nonviable
Cells, 230
11.3.10Primary Culture in Summary, 232
11.3. llPrimary Records, 232
References, 232
Suppliers, 233
. Supplementary Material*
Protocol 11.5-Su. Maximal Serial Transfer (MST) of
Human Fibroblastfrom Skin Explants
Reference
. Subculture and Cell Lines, 235
12.1 Terminology, 235
12.2 Subculture and Propagation, 235
12.2.1 Cross-contamination and
Misidentification, 238
12.2.2 Mycoplasma Contamination, 238
12.2.3 Naming a Cell Line, 238
12.2.4 Culture Age, 239
12.2.5 Finite and Continuous Cell Lines, 239
12.3 Choosing a Cell Line, 239
12.4 Routine Maintenance, 240
12.4.1 Significance of Cell Morphology, 240
12.4.2 Replacement of Medium, 241
12.4.3 Standard Feeding Protocol, 242
Protocol 12.1. Feeding a Monolayer Culture in
Flasks, 242
Protocol 12.2. Feeding A Monolayer Culture in
Plates or Dishes, 243
12.5 Subculture, 244
12.5.1 Criteria for Subculture, 244
12.5.2 Typical Subculture Protocol for Cells
Grown as a Monolayer, 246
Protocol 12.3. Subculture of Monolayer Cells, 246
12.5.3 Growth Cycle and Split Ratios, 249
Protocol 12.4. Growth Cycle Test for New
Cells, 249
12.5.4 Propagation in Suspension, 252
12.5.5 Subculture of Cells Growing in
Suspension, 252
Protocol 12.5. Subculture of Suspension Cells, 253
12.5.6 Standardization of Culture
Conditions, 254
12.5.7 Use of Antibiotics, 254
12.5.8 Maintenance Records, 255
References, 256
Suppliers, 258
13. Authentication and Validation, 259
13.1 Authentication of Cell Lines, 259
13.1.1 Misidentification and Cross-
contamination, 259
13.1.2 Causes of Misidentification and Cross-
contamination, 263
13.1.3 Avoiding Misidentification and
Cross-contamination, 263
13.1.4 Techniques for Cell Line
Authentication, 264
Minireview 13.1. DNA Profiling and Its Role in
Cell Line Authentication 265
13.1.5 DNA Profiling, 267
Protocol 13.1. DNA STR Profiling of Cell Lines, 267
13.1.6 Determining Animal Cell Species through
DNA Barcoding, 270
13.1.7 Isoenzyme Analysis, 270
Protocol 13.2. COl Barcoding of Animal Cells, 270
Protocol 13.3. Isoenzyme Analysis, 276
13.1.8 Chromosome Content, 278
Protocol 13.4. Chromosome Preparations, 278
13.1.9 Chromosome Banding, 280
13.1.10Chromosome Analysis, 281
13.1.11The Need for Authentication, 281
13.2 Validation, 281
13.2.1 Provenance, 282
13.2.2 Microbial Contamination, 283
13.2.3 Procedures, 283
13.3 Quality Assurance, 283
13.3.1 Cell Lines, 283
13.3.2 Media and Reagents, 284
13.3.3 CultureVessels, 284
13.3.4 Equipment, 284
13.3.5 Facilities, 284
References, 284
Suppliers, 287
14. Microbial Contamination. 289
14.1 Sources of Contamination, 289
14.1.1 Operator Technique, 289
xii
CONTENTS
14.1.2 Environment, 289
14.1.3 Use and Maintenance ofBSCs, 293
14.1.4 Humid Incubators, 293
14.1.5 Cold Stores, 293
14.1.6 Sterile Materials, 293
14.1.7 Imported Cell Lines and Biopsies, 294
14.1.8 Quarantine, 294
14.2 Types of Microbial Contamination, 294
14.3 Monitoring for Contamination, 294
14.3.1 Visible Microbial Contamination, 295
14.3.2 Mycoplasma, 295
14.3.3 Fluorescence Staining for
Mycoplasma, 295
Protocol 14.1. Fluorescence Detection of
Mycoplasma, 297
14.3.4 PCR for Mycoplasma, 297
Protocol 14.2. Detection of Mycoplasma by
PCR, 298
14.3.5 Alternative Methods for Detecting
Mycoplasma, 301
Protocol 14.3. Eradication of Microbial
Contamination, 302
14.3.6 Mycoplasma Detection Services, 302
14.3.7 Viral Contamination, 302
14.4 Disposal of Contaminated Cultures, 302
14.5 Eradication of Contamination, 302
14.5.1 Bacteria, Fungi, andYeasts, 302
Protocol 14.4. Eradication of Mycoplasma
Contamination, 303
14.5.2 Eradication of Mycoplasma, 303
14.5.3 Eradication of Viral Contamination, 304
14.5.4 Persistent Contamination, 304
14.6 Cross-contamination, 305
14.7 Conclusions, 305
References, 305
Suppliers, 306
15. Cryopreservation and Banking, 307
15.1 Rationale for Freezing, 307
15.2 Considerations before Cryopreservation, 307
15.2.1 Validation, 307
15.2.2 When to Freeze, 308
15.3 Principles of Cryopreservation, 308
15.3.1 Theoretical Background to Cell
Freezing, 308
15.3.2 Cell Concentration, 308
15.3.3 Freezing Medium, 308
15.3.4 Cooling Rate, 309
15.3.5 Cryovials, 311
15.3.6 Cryofreezers, 313
15.3.7 Freezing Cultured Cells, 315
Protocol 15.1. Freezing Cells, 315
15.3.8 Freezer Records, 318
15.3.9 Thawing Stored Cryovials, 318
Protocol 15.2. Thawing Frozen Cells, 319
15.3.10 Freezing Flasks, 320
15.3.11 Vitrification, 320
15.4 Design and Control of Freezer Stocks, 320
15.4.1 Inventory Control, Distribution, and
Usage, 320
15.4.2 Serial Replacement of Culture Stock, 322
15.5 Cell Banks, 322
15.6 Transporting Cells, 323
15.6.1 Frozen Cryovials, 323
15.6.2 Living Cultures, 324
References, 324
Suppliers, 326
16. Cloning and Selection, 327
16.1 Cell Cloning, 327
Protocol 16.1. Dilution Cloning, 328
16.2 Stimulation of Plating Efficiency, 331
16.2.1 Conditions that Improve Clonal
Growth, 331
16.2.2 Conditioned Medium, 332
Protocol 16.2. Preparation of Conditioned
Medium, 333
16.2.3 Feeder Layers, 333
Protocol 16.3. Preparation of Feeder Layers, 334
16.3 Suspension Cloning, 334
Protocol 16.4. Cloning in Agar, 334
Protocol 16.5. Cloning in Methocel, 337
16.4 Isolation of Ciones, 338
Protocol 16.6. Isolation of Ciones with Cloning
Rings, 338
16.4.1 Other Isolation Techniques for
Monolayer Ciones, 340
16.4.2 Suspension Ciones, 340
Protocol 16.7. Isolation of Suspension Ciones, 340
16.5 Replica Plating, 341
16.6 Selective Media and Inhibitors, 341
16.7 Interaction with Substrate, 342
16.7.1 Selective Adhesion, 342
16.7.2 Selective Detachment, 342
16.7.3 Nature of Substrate, 342
16.7.4 Selective Feeder Layers, 342
16.7.5 Selection by Semisolid Media, 342
References, 343
Suppliers, 345
16. Supplementary Material*
Clonal Isolation
Protocol 16.7-Su. Isolating Cell Colonies by
Irradiation
CONTENTS #• XÜi
Derivation of Drug-Resistant Cell Strains
Protocol 16.9-Su, Methotrexate Resistance and
DHFR Amplification
References
17. Cell Sorting, 347
17.1 Cell Density and Isopyknic
Sedimentation, 347
Protocol 17. 1. Cell Separation by Centrifugation on
a Density Gradient, 347
17.2 Cell Size and Sedimentation Velocity, 350
17.2.1 Unit Gravity Sedimentation, 350
17.2.2 Centrifugal Elutriation, 351
17.3 Antibody-Based Techniques, 352
17.3.1 Magnetic Sorting, 352
Protocol 17.2. Magnet-Activated Cell Sorting
(MACS), 353
17.3.2 Fluorescence-Activated Cell Sorting, 354
17.4 Beginner s Approach to Cell Separation, 356
References, 356
Suppliers, 357
18. Cell Line Characterizatlon, 359
18.1 The Need for Characterization, 359
18.2 Genotypic Characterization, 359
18.2.1 Authentication, 360
18.2.2 Species Identification, 360
18.2.3 Transformation, 360
18.3 Phenotypic Characterization, 360
18.3.1 Lineage orTissue Markers, 360
18.3.2 Unique Markers, 362
18.4 Cell Morphology, 362
18.4.1 Microscopy, 368
Protocol 18.1. Using an Inverted Microscope, 369
18.4.2 Staining, 369
Protocol 18.2. Staining with Giemsa, 370
Protocol 18.3. Staining with CrystalViolet, 370
18.4.3 CultureVessels for Cytology:
Monolayer Cultures, 371
18.4.4 Preparation of Suspension Culture for
Cytology, 371
Protocol 18.4. Preparation of Suspension Cells for
Cytology by Cytocentrifuge, 372
18.4.5 Photomicrography, 372
Protocol 18.5. Digital Photography On A
Microscope, 372
18.4.6 Live-Cell Imaging, 373
18.4.7 Confocal Microscopy, 374
18.5 Antigenic Markers, 374
18.5.1 Immunostaining, 375
Protocol 18.6. Indirect Immunofluorescence, 376
18.5.2 Immunoanalysis, 376
18.6 Time-Lapse Recording, 377
Protocol 18.7. Time-Lapse Videorecording, 377
18.7 Cell Cycle, 378
18.7.1 Cell Separation, 379
18.7.2 Blockade, 379
References, 379
Suppliers, 381
19. Differentiation, 383
19.1 Expression of the In Vivo Phenotype, 383
19.1.1 DedifFerentiation, 384
19.1.2 Lineage Selection, 384
19.2 Stages of Differentiation, 384
19.3 Proliferation and Differentiation, 384
19.4 Commitment and Lineage, 385
19.5 Stern Cell Plasticity, 386
19.6 Markers of Differentiation, 386
19.7 Induction of Differentiation, 387
19.7.1 Cell Interaction, 387
19.7.2 Systemic Factors, 389
19.7.3 Cell—Matrix Interactions, 392
19.7.4 Polarity and Cell Shape, 392
19.7.5 Dynamic Stress, 393
19.7.6 Oxygen Tension, 393
19.8 Differentiation and Malignancy, 393
19.9 Practical Aspects, 393
References, 394
20. Ihree-Dlmenslonal Culture 401
20.1 Cell Interaction and Phenotypic
Expression, 401
20.1.1 Advantages and Limitations of
3D Culture, 402
20.1.2 Choice of Models, 403
Minireview 20.1. Advances in Technologies Enabling
Three-Dimensional Cell Culture and the Formation of
Tissue-like Architecture In Vitro, 403
20.2 Organ Culture, 407
20.2.1 Gas and Nutrient Exchange, 407
20.2.2 Structural Integrity, 408
20.2.3 Growth and Differentiation, 408
20.2.4 Limitations of Organ Culture, 408
20.2.5 Types of Organ Culture, 408
Protocol 20.1. Organ Culture, 408
20.3 Histotypic Culture, 409
20.3.1 Gel and Sponge Techniques, 409
20.3.2 Hollow Fibers, 410
20.3.3 Spheroids, 410
Protocol 20.2. 3D Culture in Spheroids, 410
XiV ¦« CONTENTS
20.3.4 Hanging-Drop Culture, 412
20.3.5 Microcarriers, 413
20.3.6 Organoids, 413
20.3.7 Rotating-Chamber Systems, 413
20.3.8 Immobilization of Living Cells in
Alginate, 415
20.3.9 Filter Well Inserts, 415
Protocol 20.3. Filter Well Inserts, 415
Protocol 20.4. Neuronal Aggregates, 417
20.3.10Cultures of Neuronal Aggregates, 418
20.4 Organotypic Culture, 418
20.4.1 Tissue Equivalents, 418
20.4.2 Tissue Engineering, 419
20.5 Imaging Cells in 3D Constructs, 420
References, 420
Suppliers, 425
21. Scale-up and Automation. 427
21.1 Scale-up in Suspension, 428
Protocol 21.1. Stirred 4-L Batch Suspension
Culture, 428
21.1.1 Continuous and Batch Culture, 430
21.1.2 Single-use Bioreactors, 430
21.1.3 Scale and Complexity, 431
21.1.4 Mixing and Aeration, 432
21.2 Scale-up in Monolayer, 436
21.2.1 Multisurface Propagators, 436
Protocol 21.2. Nunc Cell Factory, 436
21.2.2 Roller Culture, 439
Protocol 21.3. Roller Bottie Culture, 439
21.2.3 Microcarriers, 440
Protocol 21.4. Microcarriers, 441
21.2.4 Large Microcarriers, 443
21.2.5 Perfused Monolayer Culture, 443
21.3 Process Control, 444
21.4 Large-Scale Processes, 445
Minireview 21.1. Culture Scale-up and
Bioreactors, 445
21.5 Automation, 448
21.5.1 Robotic Cell Culture, 448
21.5.2 High-Throughput Screening (HTS), 450
21.5.3 HTS in Three Dimensions, 450
Minireview 21.2. Microfluidic Cell Culture, 450
References, 453
Suppliers, 454
22. Senescence, Immortalization, and
Transformation, 455
22.1 Role in Cell Line Characterization, 455
22.2 Genetic Instability and Heterogeneity, 457
22.2.1 Genetic Instability, 457
22.2.2 Chromosomal Aberrations, 457
22.3 Immortalization, 458
22.3.1 Control of Senescence, 458
22.3.2 Immortalization withViral Genes, 459
Minireview 22.1. Senescence and
Immortalization, 459
22.3.3 Immortalization of Human
Fibroblasts, 462
Protocol 22.1. Fibroblast Immortalization, 462
22.3.4 Telomerase-Induced Immortalization, 465
Protocol 22.2. Immortalization of Human Stern and
Primary Cells by Telomerase, 465
22.3.5 Alternative Immortalization
Strategies, 468
22.4 Aberrant Growth Control, 468
22.4.1 Anchorage Independence, 468
22.4.2 Contact Inhibition, 469
Protocol 22.3. Density Limitation of Cell
Proliferation, 470
22.4.3 Serum Dependence, 471
22.4.4 Oncogenes, 471
22.5 Tumorigenicity, 471
22.5.1 Malignancy, 471
22.5.2 Tumor Transplantation, 472
22.5.3 Invasiveness, 472
22.5.4 Angiogenesis, 473
Protocol 22.4. In Vitro Angiogenesis Assay, 473
22.5.5 Plasminogen Activator, 476
References, 476
Supplier, 481
23. Quantitation, 483
23.1 Cell Counting, 483
23.1.1 Hemocytometer, 483
Protocol 23.1. Cell Counting by
Hemocytometer, 483
23.1.2 Electronic Counting, 487
Protocol 23.2. Electronic Cell Counting by Electrical
Resistance, 488
23.1.3 Stained Monolayers, 491
23.1.4 Flow Cytometry, 491
23.2 Cell Weight and Packed Cell Volume, 491
23.3 DNA Content, 493
23.4 Protein, 493
23.5 Rates of Synthesis, 493
23.5.1 DNA Synthesis, 493
23.5.2 Protein Synthesis, 493
23.6 Preparation of Samples for Enzyme Assay and
Immunoassay, 493
23.7 Cytometry, 494
23.7.1 In situ Labeling, 494
23.7.2 Flow Cytometry, 494
CONTENTS ^5* XV
23.8 Replicate Sampling, 494
23.8.1 Data Acquisition, 494
23.8.2 Data Analysis, 494
23.9 Cell Proliferation, 495
23.9.1 Experimental Design, 495
23.9.2 Growth Cycle, 495
Protocol 23.7. Growth Curve with a Monolayer in
Flasks, 497
Protocol 23.8. Growth Curve with a Monolayer in
Multiwell Plates, 498
23.9.3 Analysis of Monolayer Growth
Curves, 499
23.9.4 Medium Volume, Cell Concentration,
and Cell Density, 501
29.9.5 Suspension Cultures, 501
Protocol 23.9. Growth Curve with Cells in
Suspension, 501
29.9.6 Phases of the Growth Cycle, 502
29.9.7 Derivatives from the Growth Curve, 502
23.10 Plating Efficiency, 503
Protocol 23.10. Determination of Plating
Efficiency, 504
23.10.1 Analysis of Colony Formation, 506
23.10.2 Automatic Colony Counting, 506
23.11 Labeling Index, 507
23.11.1 Growth Fraction, 507
23.11.2 Mitotic Index, 509
23.11.3 Division Index, 509
23.12 Autoradiography, 509
23.13 Cell Cycle Time, 510
23.14 Cell Migration, 510
References, 510
Suppliers, 511
23 Supplementary Material*
DNA Content
Protocol 23.3-Su. DNA Estimation by Hoechst
33258
Bradford Assay
Protocol 23.4-Su. Protein Estimation by the Bradford
Method
DNA Synthesis
Protocol 23.5-Su. Estimation ofDNA Synthesis by
[3H] Thymidine Incorporation
Protein Synthesis
Protocol 23.6-Su. Estimation of Protein Synthesis by
Incorporation of [3H] Leucine
Determination of Labeling Index and Growth
Fraction by Autoradiography
Protocol 23.1 i-Su. Labeling Index with
[3H] Thymidine
Protocol 23.12-Su. Determination of Growth
Fraction
Protocol 23.13-Su. Microautoradiography
References
Suppliers
24. Cytotoxicity, 513
24.1 Viability, Toxicity, and Survival, 513
24.2 In Vitro Limitations, 514
24.2.1 Pharmacokinetics, 514
24.2.2 Metabolism, 514
24.2.3 Tissue and Systemic Responses, 514
24.3 Nature of the Assay, 515
24.3.1 Viability, 515
Protocol 24.1. Estimation of Viability by Dye
Exclusion, 515
Protocol 24.2. Estimation of Viability by Dye
Uptake, 516
24.3.2 Survival, 517
Protocol 24.3. Clonogenic Assay forAttached
Cells, 517
24.3.3 Assays Based on Cell Proliferation, 520
24.3.4 Metabolie Cytotoxicity Assays, 520
24.3.5 Microtitration Assays, 521
Protocol 24.4. MTT-Based Cytotoxicity Assay, 521
24.3.6 Comparison of Microtitration
with Clonogenic Survival, 524
24.3.7 Drug Interaction, 525
24.4 Applications of Cytotoxicity Assays, 525
24.4.1 Anticancer Drug Screening, 525
24.4.2 Predictive DrugTesting for Tumors, 525
24.4.3 Testing Pharmaceuticals, 526
24.5 Genotoxicity, 526
24.5.1 Mutagenesis Assay by Sister Chromatid
Exchange, 526
24.5.2 Carcinogenicity, 526
24.6 Inflammation, 526
References, 527
Suppliers, 529
24. Supplementary Material*
Assaying Mutagenesis by Sister Chromatid
Exchange
Protocol 24.5-Su. Sister Chromatid Exchange
References
Suppliers
25. Culture of Specific Cell Types, 531
25.1 Cell Culture of Specialized Cells, 534
25.2 Epithelial Cells, 534
25.2.1 Epidermis, 534
xvi
CONTENTS
25.2.2 Cornea, 535
25.2.3 Breast, 535
25.2.4 Cervix, 536
25.2.5 GastrointestinalTract, 537
25.2.6 Liver, 537
25.2.7 HepaRG Human Hepatocytes, 537
25.2.8 Pancreas, 537
25.2.9 Bronchial andTracheal Epithelium 537
25.2.10 Oral Epithelium, 538
25.2.11 Prostate, 538
25.3 Mesenchymal Cells, 538
25.3.1 ConnectiveTissue, 538
25.3.2 Adipose Tissue, 539
25.3.3 Muscle, 539
25.3.4 Cartilage, 540
25.3.5 Bone, 540
25.3.6 Endothelium 540
25.4 Neuroectodermal Cells, 541
25.4.1 Neurons, 541
25.4.2 Glial Cells, 541
25.4.3 Endocrine Cells, 542
25.4.4 Melanocytes, 543
25.5 Hematopoietic Cells, 543
25.5.1 Erythroid Cells, 543
25.5.2 Myeloid Cells and Macrophages, 544
25.5.3 Lymphoid Cells, 544
25.6 Production of Monoclonal Antibodies, 544
25.7 Gonads, 544
25.7.1 Ovary, 544
25.7.2 Testis, 544
25.8 Culture of Cells from Poikilotherms, 546
25.8.1 Fish Cells, 546
25.8.2 Insect Cells, 546
25.9 Tumor Cell Culture, 546
25.10 Sampling, 547
25.10.1 Selection of Representative Cells, 547
25.10.2 Preservation of Tissue by Freezing, 548
Protocol 25.27. Freezing Biopsies, 548
25.11 Disaggregation, 548
25.12 Primary Culture, 548
25.13 Selective Culture of Tumor Cells, 549
25.13.1 Selective Media, 549
25.13.2 Confluent Feeder Layers, 549
Protocol 25.28. Growth on Confluent Feeder
Layers, 551
25.13.3 Suspension Cloning, 551
25.13.4 Xenografts, 552
25.14 Development of Cell Lines, 552
25.14.1 Subculture of Primary Tumor
Cultures, 552
25.14.2 Continuous Cell Lines, 552
25.15 Characterization of Tumor Cell Cultures, 553
25.15.1 Heterogeneity of Tumor Cultures, 553
25.15.2 Histotypic Culture, 553
25.16 Specific Tumor Types, 554
25.16.1 Breast, 554
25.16.2 Lung, 554
25.16.3 Stomach, 554
25.16.4 Colon, 554
25.16.5 Pancreas, 556
25.16.6 Ovary, 556
25.16.7 Prostate, 556
25.16.8 Bladder, 557
25.16.9 Skin, 557
25.16.10 Cervix, 558
25.16.11 Glioma, 558
25.16.12 Neuroblastoma, 558
25.16.13 Seminoma, 558
25.16.14 Lymphoma and Leukemia, 558
References, 559
Suppliers, 569
25. Supplementär* Material
Protocol 25.1-Su. Epidermal Keratinocytes
Protocol 25.2-Su. Corneal Epithelial Cells
Protocol 25.3-Su. Preparation ofMammary
Epithelial Cells from Reduction
Mammoplasty Specimens
Protocol 25.4-Su. Cervical Epithelium
Protocol 25.5-Su. Isolation and Culture of
Colonic Crypts
Protocol 25.6-Su. Isolation of Rat Hepatocytes
Protocol 25.7-Su. Purifiation of HepaRG Human
Hepatocytes
Protocol 25.8-Su. Pancreatic Epithelium
Protocol 25.9-Su. Bronchial and Tracheal Epithelium
Protocol 25.10-Su. Oral Keratinocytes
Protocol 25.11-Su. Rat Prostatic Epithelial Cells
Protocol 25.12-Su. Primary Culture of
Adipose Cells
Protocol 25.13-Su. Isolation and Culture of
Smooth-Muscle Cells
Protocol 25.14-Su. Culture of Myohlasts from Adult
Skeletal Muscle
Protocol 25.15-Su. Single-Myofiber Culture from
Skeletal Muscle
Protocol 25.16-Su. Culturing Chondrocytes in
Alginate Beads
Protocol 25.17-Su. Isolation and Culture ofVascular
Endothelial Cells
Protocol 25.18-Su. Cerebellar Granule Cells
Protocol 25.19-Su. Primary Culture of Human
Astrocytes
Protocol 25.20-Su. Rat Olfactory Ensheathing Cells
Protocol 25.21 -Su. Culture of Melanocytes
Protocol 25.22-Su. Preparation of Lymphocytes
Protocol 25.23-Su. PHA Stimulation of Lymphocytes
Protocol 25.24-Su. Production Of Monoclonal
Antibodies ByThe B-Cell Targeting Technique (Bct)
CONTENTS XVÜ
Protocol 25.25-Su. Production Of Monoclonal
Antibodies ByThe Stereospecific Targeting (Sst)
Technique
Protocol 25.26-Su. Propagation oflnsect Cells
Protocol 25.29-Su. Culture of Mammary Tumor Cells
Protocol 25.30-Su. Culture of Colorectal Tumors
Protocol 25.31-Su. Establishment of Continuous
Cell Lines from Leukemia /Lymphoma
References
Suppliers
Protocol 26.2-Su. Propagation of Mouse Embryonic
Stem Cell Lines
Protocol 26.3-Su. Derivation, Culture and
Cryopreservation of Human Embryonic Stem Cells
Protocol 26.4-Su. Cell Cultures from Zebrafish
Embryos
Culture of Amniocytes
MSC Production from Human
Protocol 26.5-Su.
Protocol 26.6-Su.
Bone Marrow
Protocol 26.7-Su.
Cells
Protocol 26.8-Su.
Protocol 26.9-Su.
Mouse Prostatic Epithelial Stem
Dental Epithelial Stem Cells
Reprogramming Human Dermal
Fibroblasts for the Generation of Pluripotent Stem
Cells
Protocol 26.10-Su. Human Long-Term Culture-
Initiating Cell (LTC-IC) Assay
References
Suppliers
26. Stein Cells, 571
26.1 Embryonic Stem Cells, 571
26.1.1 Derivation of Mouse Embryonic Stem
Cells, 571
26.1.2 Subculture and Propagation of Mouse
Embryonic Stem Cells, 571
26.1.3 Primary Culture of Human Embryonic
Stem Cells, 572
26.1.4 Pluripotent Stem Cells from
Fish Embryos, 572
26.2 Germ Cells, 572
26.3 Extraembryonic Cells, 572
26.3.1 Culture of Amniocytes, 574
26.3.2 Cells from Neonates and Juveniles, 574
26.3.3 Multipotent Stem Cells from the
Adult, 575
26.3.4 MSCs from Human Bone Marrow, 575
26.3.5 Prostate Epithelial Stem Cells, 575
26.3.6 Dental Epithelial Stem Cells, 575
26.3.7 Induced Pluripotent Stem Cells, 575
26.4 Hemopoietic Stem Cells, 575
Minireview 26.1. Growth Control of Hemopoietic
Cells in Culture, 577
26.4.1 Long-Term Bone Marrow Cultures
from Mouse, 580
26.4.2 Human Long-Term Bone Marrow
Cultures, 580
26.5 Appication of Stem Cells to Regenerative
Medicine, 580
Minireview 26.2. Cell-Based Regenerative
Medicine, 580
26.6 Cancer Stem Cells, 582
Minireview 26.3. Culture of Cancer Stem
Cells, 583
References, 584
26. Supplementary Material*
Protocol 26.1-Su Derivation and Primary Culture of
Mouse Embryonic Stem Cells
11. Training Programs, 591
27.1 Objectives, 591
27.2 Preparative and Manipulative Skills, 594
Exercise 1, Sterile Pipetting and Transfer of
Fluids, 594
Exercise 2, Washing and Sterilizing Glassware, 596
Exercise 3, Preparation and Sterilization
ofWater, 596
Exercise 4, Preparation and Sterilization ofDulbecco s
Phosphate-Buffered Saline (D-PBS) without Ca2+
and Mg2+ (D-PBSA), 597
Exercise 5, Preparation of pH Standards, 598
Exercise 6, Preparation of Stock Medium from Powder
and Sterilization by Filtration, 599
27.3 Basic Cell Culture Tecbniques, 601
Exercise 7, Observation of Cultured Cells, 601
Exercise 8, Preparing Sterile Medium for Use, 602
Exercise 9, Feeding a Monolayer Culture, 604
Exercise 10, Preparation of Complete Medium from
10x Stock, 605
Exercise 11, Counting Cells by Hemocytometer and
Electronic Counter, 607
Exercise 12, Subculture of Cells Growing in
Suspension, 609
Exercise 13, Subculture of Cell Lines Growing in
Monolayer, 610
Exercise 14, Staining a Monolayer Cell Culture with
Giemsa, 613
Exercise 15, Construction and Analysis of Growth
Curve, 614
27.4 Advanced Exercises, 616
Exercise 16, Cell Line Characterization, 616
xviii * CONTENTS
Excrcisc 17, Detection of Mycoplasma, 617
Exercise 18, Cryopreservation of Cultured
Cells, 618
Exercise 19, Primary Culture, 621
Exercise 20, Cloning of Monolayer Cells, 624
27.5 Specialized Exercises, 626
References, 626
Suppliers, 626
28. Problem Solving 627
28.1 Abnormal Appearance of Cells, 627
28.2 Slow Cell Growth, 628
28.2.1 Problems Restricted toYour Own
Stock, 628
28.2.2 Problem Is More General and Other
People Are Having Difficulty, 629
28.3 Medium, 630
28.3.1 Formulation, Preparation, and
Storage, 630
28.3.2 Unstable Reagents, 631
28.3.3 Purity of Medium Constituents, 632
28.4 Substrates and Containers, 633
28.5 Microbial Contamination, 633
28.5.1 Confined to Single Use, 633
28.5.2 Widespread, 635
28.5.3 Air Supply and BSCs, 636
28.5.4 Specific Contaminants, 637
28.6 Chemical Contamination, 638
28.6.1 Glassware, 638
28.6.2 Glass Pipettes, 638
28.6.3 Water Purification, 638
28.6.4 Cryopreservatives, 638
28.6.5 Powders and Aerosols, 639
28.7 Primary Culture, 639
28.7.1 PoorTake in Primary Culture, 639
28.7.2 Wrong Cells Have Been Selected, 640
28.7.3 Contamination, 641
28.8 Differentiation, 641
28.9 Feeding, 641
28.9.1 Regulär Monolayers, 641
28.9.2 Feeding Cell Cloning, 642
28.10 Subculture, 642
28.10.1 Poor Take or Slow Growth, 642
28.10.2 Uneven Growth, 643
28.11 Cloning, 644
28.11.1 Too Few Colonies per Dish, 644
28.11.2 Too Many Colonies per Dish, 645
28.11.3 Nonrandom Distribution 645
28.12 Cross-contamination and
Misidentification, 646
28.13 Cryopreservation, 646
28.13.1 Poor Recovery, 646
28.13.2 Changed Appearance after
Cryopreservation, 647
28.13.3 Loss of Stock, 647
28.14 Cell Counting, 648
28.14.1 Counting with a Hemocytometer, 648
28.14.2 Electronic Counting by Resistance, 648
Reference, 649
Suppliers, 649
29. In Eonclusion, 651
Appendix I: Calculations and Preparation of Reagents, 653
Calculations, 653
Conversions, 653
Preparation of Reagents, 654
Appendix II: Glossary, 663
Index, 671
Supplementary Material*
Appendix III: Sources of Equipment and Material
Appendix IV: Suppliers and Other Resource
*Supplementary material is available at the following Website:
www.wiley.com/go/freshney/cellculture_7e
|
| any_adam_object | 1 |
| author | Freshney, Robert Ian 1938-2019 |
| author2 | Capes-Davis, Amanda |
| author2_role | edt |
| author2_variant | a c d acd |
| author_GND | (DE-588)1055981578 (DE-588)1112631836 |
| author_facet | Freshney, Robert Ian 1938-2019 Capes-Davis, Amanda |
| author_role | aut |
| author_sort | Freshney, Robert Ian 1938-2019 |
| author_variant | r i f ri rif |
| building | Verbundindex |
| bvnumber | BV042713640 |
| classification_rvk | WX 6600 WX 6603 |
| ctrlnum | (OCoLC)945012559 (DE-599)BVBBV042713640 |
| discipline | Biologie |
| edition | Seventh edition |
| format | Book |
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| id | DE-604.BV042713640 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T07:08:01Z |
| institution | BVB |
| isbn | 9781118873656 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-028144930 |
| oclc_num | 945012559 |
| open_access_boolean | |
| owner | DE-20 DE-703 DE-11 DE-578 DE-634 DE-19 DE-BY-UBM |
| owner_facet | DE-20 DE-703 DE-11 DE-578 DE-634 DE-19 DE-BY-UBM |
| physical | xxxviii, 684 Seiten Illustrationen, Diagramme |
| publishDate | 2016 |
| publishDateSearch | 2016 |
| publishDateSort | 2016 |
| publisher | Wiley Blackwell |
| record_format | marc |
| spelling | Freshney, Robert Ian 1938-2019 Verfasser (DE-588)1055981578 aut Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney ; reviewing editors: Amanda Capes-Davis, Carl Gregory, Stefan Przyborski Seventh edition Hoboken, New Jersey Wiley Blackwell [2016] © 2016 xxxviii, 684 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Tiere (DE-588)4060087-7 gnd rswk-swf Gewebekultur (DE-588)4157245-2 gnd rswk-swf Zellkultur (DE-588)4067547-6 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Tierzelle (DE-588)4185509-7 gnd rswk-swf Zellkultur (DE-588)4067547-6 s Tiere (DE-588)4060087-7 s Methode (DE-588)4038971-6 s DE-604 Tierzelle (DE-588)4185509-7 s 1\p DE-604 Gewebekultur (DE-588)4157245-2 s 2\p DE-604 Capes-Davis, Amanda (DE-588)1112631836 edt HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028144930&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Freshney, Robert Ian 1938-2019 Culture of animal cells a manual of basic technique and specialized applications Tiere (DE-588)4060087-7 gnd Gewebekultur (DE-588)4157245-2 gnd Zellkultur (DE-588)4067547-6 gnd Methode (DE-588)4038971-6 gnd Tierzelle (DE-588)4185509-7 gnd |
| subject_GND | (DE-588)4060087-7 (DE-588)4157245-2 (DE-588)4067547-6 (DE-588)4038971-6 (DE-588)4185509-7 |
| title | Culture of animal cells a manual of basic technique and specialized applications |
| title_auth | Culture of animal cells a manual of basic technique and specialized applications |
| title_exact_search | Culture of animal cells a manual of basic technique and specialized applications |
| title_full | Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney ; reviewing editors: Amanda Capes-Davis, Carl Gregory, Stefan Przyborski |
| title_fullStr | Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney ; reviewing editors: Amanda Capes-Davis, Carl Gregory, Stefan Przyborski |
| title_full_unstemmed | Culture of animal cells a manual of basic technique and specialized applications R. Ian Freshney ; reviewing editors: Amanda Capes-Davis, Carl Gregory, Stefan Przyborski |
| title_short | Culture of animal cells |
| title_sort | culture of animal cells a manual of basic technique and specialized applications |
| title_sub | a manual of basic technique and specialized applications |
| topic | Tiere (DE-588)4060087-7 gnd Gewebekultur (DE-588)4157245-2 gnd Zellkultur (DE-588)4067547-6 gnd Methode (DE-588)4038971-6 gnd Tierzelle (DE-588)4185509-7 gnd |
| topic_facet | Tiere Gewebekultur Zellkultur Methode Tierzelle |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028144930&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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