Freshney's culture of animal cells: a manual of basic technique and specialized applications
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| Format: | Buch |
| Sprache: | English |
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Hoboken, NJ, USA
Wiley Blackwell
[2021]
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| Ausgabe: | Eighth edition |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | "A companion website with additional resources is available at: www.wiley.com/go/freshney/cellculture8." - Rückseite Cover |
| Beschreibung: | xxix, 796 Seiten Illustrationen, Diagramme |
| ISBN: | 9781119513018 |
Internformat
MARC
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| 245 | 1 | 0 | |a Freshney's culture of animal cells |b a manual of basic technique and specialized applications |c Amanda Capes-Davis, R. Ian Freshney ; reviewing editors: Robert J Geraghty, Raymond W. Nims |
| 246 | 1 | 3 | |a Culture of animal cells |
| 250 | |a Eighth edition | ||
| 264 | 1 | |a Hoboken, NJ, USA |b Wiley Blackwell |c [2021] | |
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| 300 | |a xxix, 796 Seiten |b Illustrationen, Diagramme | ||
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Datensatz im Suchindex
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| adam_text | Foreword, xix Acknowledgments, xxi Abbreviations, xxiii Book Navigation, xxix PART I Understanding Cel! Culture, 1 1. Introduction, 3 1.1 Terminology, 3 1.1.1 Tissue Culture and Cell Culture, 3 1.1.2 Sources of Terminology, 3 1.2 Historical Development, 4 1.2.1 Substrates and Media, 4 1.2.2 Primary Cultures and Cell Lines, 5 1.2.3 Organ, Organotypic, and Organoid Culture, 7 1.3 Applications, 12 1.4 Advantages of Tissue Culture, 13 1.4.1 Environmental Control, 13 1.4.2 Homogeneity and Characterization, 14 1.4.3 Economy, Scale, and Automation, 14 1.4.4 Replacement of In Vivo Models, 14 1.5 Limitations of Tissue Culture, 15 1.5.1 Quality and Expertise, 15 1.5.2 Quantity and Cost, 16 1.5.3 Limited Species and Cell Types, 17 1.5.4 Limited Understanding of the Cell and its Microenvironment, 17 References, 18 l. Biology of Cultured Culls, 33 2.1 2.2 The Culture Environment, 23 Cell Adhesion, 23 2.2.1 Intercellular Junctions, 25 2.2.2 Cell Adhesion Molecules, 25 2.2.3 Cytoskeleton, 26 2.2.4 Extracellular Matrix (ECM), 27 2.2.5 Cell Motility, 27 2.3 Cell Division, 28 2.3.1 Cell Cycle, 28 2.3.2 Control of the Cell Cycle, 29 2.4 Cell Fate, 30 2.4.1 Embryonic Lineages, 30 2.4.2 Stem Cells and Potency, 31 2.4.3 Differentiation, 31 2.4.4 Control of Potency and Differentiation, 32 2.4.5 Lineage Commitment, 33 2.4.6 Lineage Plasticity, 35 2.5 Cell Death, 35 References, 36 3. Origin and Mtion of Cultured Cells, 39 3.1 3.2 Origin of Cultured Cells, 39 3.1.1 Sample Origin, 39 3.1.2 Disease Origin, 40 Evolution of Cell Lines, 40 3.2.1 Phases of Cell Cultivation, 40 3.2.2 Clonal Evolution, 41
VII
VIII Contents 3.3 Changes in Genotype, 43 3.3.1 3.3.2 3.4 Phenotypic Variation, 46 Phenotype and Culture Conditions, 47 5.3 Senescence and Immortalization, 48 Minireview М3.1 Senescence and 3.6 Intrinsic Control of Senescence, 50 Extrinsic Control of Senescence, 50 5.4 3.6.1 3.6.2 3.6.3 Characteristics of Transformation, 50 Aberrant Growth Control, 52 Tumorigenicity and Malignancy, 55 5.5 3.7 Conclusions: Origin and Evolution, 58 References, 58 РАШИ ^908298^334812 ՛ . V. Requirements, 63 4. laboratory Design and Layout, 66 4.1 4.2 Sterile Handling Area, 75 Incubation Area, 76 Quarantine Area, 77 Preparation Area, 77 Washup Area, 77 Storage Area, 78 6. Safety and Bioethics, 111 6.1 6.2 6.3 5.1.4 6.4 Needlestick and Sharps Injuries, 118 Hazardous Substances, 118 Asphyxia and Explosion, 119 Burns and Frostbite, 120 Fire, 120 Equipment Hazards, 120 Biosafety, 121 6.3.6 6.3.7 6. Equipment and Materials, 86 Biological Safety Cabinet (BSC), 85 BSC Services and Consumables, 88 Sterile Liquid Handling Equipment, 90 Centrifuge, 94 Hazards in Tissue Culture Laboratories, 117 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 References, 83 5.1.1 5.1.2 5.1.3 Risk Assessment, 111 Safety Regulations, 113 Training, 116 Ergonomics, 117 6.2.1 6.2.2 6.2.3 6.2.4 6.2.5 6.2.6 Contingency Plans and Priorities, 81 Equipment Monitoring and Alarms, 83 5.1 Sterile Handling Area Equipment, 85 Laboratory Safety, 111 6.1.1 6.1.2 6.1.3 6.1.4 Disaster and Contingency Planning, 80 4.3.1 4.3.2 Cold Storage Equipment, 107 References, 109 Layout of Laboratory Areas, 74 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.3 General Design
Considerations, 65 User Requirements, 67 Regulatory Requirements, 70 Engineering Requirements, 71 Water Purification Systems, 104 Preparation Equipment, 104 Washup Equipment, 105 Sterilization Equipment, 106 5.5.1 Refrigerators and Freezers, 107 5.5.2 Cryofreezers, 108 5.5.3 Rate-Controlled Freezer, 108 Suppliers, 108 Design Requirements, 65 4.1.1 4.1.2 4.1.3 4.1.4 Incubators, 100 Incubator Accessories, 103 Water Baths, 103 Preparation and Washup Equipment, 104 5.4.1 5.4.2 5.4.3 5.4.4 Transformation, 50 Microscopes, 97 Cameras, 98 Computer and Monitor, 98 Cell Counting and Analysis Equipment, 99 Incubation Equipment, 99 5.3.1 5.3.2 5.3.3 Immortalization, 48 3.5.1 3.5.2 Imaging and Analysis Equipment, 97 5.2.1 5.2.2 5.2.3 5.2.4 Changes in Phenotype, 46 3.4.1 3.4.2 3.5 5.2 Chromosomal Aberrations, 43 Genomic Variation, 44 Source of Biohazard Risk, 121 Biohazard Risk Groups, 122 Biological Containment Levels, 123 Containment Equipment, 123 Decontamination and Fumigation, 128 Waste Disposal and Disinfectants, 128 Genetically Modified Organisms (GMOs), 129 Bioethics, 129 6.4.1 Ethical Use of Animal Tissue,130 6.4.2 Ethical Use of Human Tissue,130 6.4.3 Donor Consent, 131 Suppliers, 132 References, 132
Contents 8.3.3 8.3.4 8.3.5 8.3.6 7. Reproducibility and Good Cell Colture Practice, 137 7.1 Reproducibility, 137 7.1.1 7.1.2 7.1.3 7.2 7.2.2 7.2.3 7.4.2 7.5.2 7.5.3 7.5.4 7.5.5 References, 173 Standard Operating Procedures (SOPs), 148 Media and Reagents, 149 Culture Vessels, 149 Equipment, 149 Facilities, 149 9. Defined Media and Supplements, 177 9.1 9.2 pH, 177 Buffering, 178 Carbon Dioxide (C02) and Sodium Bicarbonate (NaHC03), 178 9.2.4 Oxygen, 179 Minireview M9A Hypoxic Cell Culture, 180 9.2.5 Temperature, 183 9.2.6 Osmolality, 184 9.2.7 Viscosity, 185 9.2.8 Surface Tension and Foaming, 185 Replicate Sampling, 150 References, 151 ^ Requirements, 155 9.3 9.4 8.2 8.2.1 8.2.2 8.3 9.5 Substrate Treatments, 159 8.3.1 8.3.2 9.6 Amino Acids, 188 Vitamins, 188 Inorganic Salts, 188 Glucose, 188 Other Components, 188 Serum, 189 9.5.1 9.5.2 9.5.3 9.5.4 9.5.5 9.5.6 Common Substrate Materials, 158 Alternative Substrate Materials, 158 Substrate Conditioning, 160 Extracellular Matrix (ECM) Coatings, 160 Protocol P8. Í Application of Matrigel Coatings, 1 60 Balanced Salt Solutions, 185 Media Formulations, 186 9.4.1 9.4.2 9.4.3 9.4.4 9.4.5 R. Culture Vessels and Substrates, 157 Attachment and Growth Requirements, 157 Substrate Materials, 158 Medium Development, 177 Physicochemical Properties, 177 9.2.1 9.2.2 9.2.3 7.6.1 Experimental Design, 150 7.6.2 Samples and Data, 150 Suppliers, 151 8.1 Application-Specific Vessels, 170 8.6.1 Imaging, 170 8.6.2 Suspension Culture, 171 8.6.3 Scaffold-Free 3D Culture, 171 8.6.4 Permeable Supports, 172 8.6.5 Scaffold-Based 3D Culture, 172 Suppliers,
172 Provenance Information, 145 Reporting for Publication, 146 Testing for Microbial Contamination, 147 Testing for Authenticity, 147 Cell Yield, 166 Multiwell Plates, 166 Flasks and Petri Dishes, 166 Multilayer Flasks, 166 Lids and Venting, 167 Uneven Growth, 169 Cost, 169 Quality Assurance (QA), 148 7.5.1 7.6 8.6 Validation Testing, 146 7.4.1 7.5 Good Cell Culture Practice (GCCP), 142 Good Laboratory Practice (GLP), 142 Good Manufacturing Practice (GMP), 143 Feeder Layers, 163 Choice of Culture Vessel, 164 8.5.1 8.5.2 8.5.3 8.5.4 8.5.5 8.5.6 8.5.7 Cell Line Provenance, 145 7.3.1 7.3.2 7.4 8.3.7 8.4 8.5 Good Practice Requirements, 141 7.2.1 7.3 Terminology: Reproducible Research, 137 Causes of Irreproducible Research, 138 Solutions to Irreproducible Research, 139 Collagen and Gelatin, 161 ECM Mimetic Treatments, 161 Polymer Coatings, 161 Non-Adhesive Substrates and Patterning, 163 Other Surface Treatments, 163 Protein, 189 Hormones and Growth Factors, 190 Nutrients and Metabolites, 190 Lipids, 190 Trace Elements, 190 Inhibitors, 191 Other Media Supplements, 191 9.6.1 9.6.2 Conditioned Medium, 191 Antibiotics, 191 IX
x Contents 9.7 Choice of Complete Medium, 191 9.7.1 9.7.2 9.7.3 9.8 Serum Testing, 193 Serum Batch Reservation, 193 Serum Treatment, 193 11.4 Other Laboratory Apparatus, 229 11.4.1 11.4.2 11.5 Water, 229 11.5.1 11.5.2 Storage of Medium and Serum, 194 Suppliers, 194 References, 194 Cleaning and Packaging, 229 Sterilization or Disinfection, 229 Water Purity, 229 Purification Methods, 230 Protocol P1Í.3 Preparation and Sterilization of Ultrapure Water (UPW), 230 11.5.3 10. Serum-Free Media, 199 10.1 10.4.3 10.5 10.6 Disadvantages of Serum, 199 Advantages of Serum-Free Media, 200 Disadvantages of Serum-Free Media, 201 Hormones and Growth Factors, 203 Antioxidants, Vitamins, and Lipids, 208 Other Supplements for Serum-Free Medium, 208 10.7 10.8 10.9 11.6.2 Basal and Complete Media, 234 Protocol PI 1.5 Preparation of Medium from Powder, 235 Protocol PI 1.6 Preparation of Medium from Юх Concentrate, 236 Protocol PI 1.7 Preparation of Medium from lx Stock, 237 11.6.3 11.7 Serum, 238 Sterile Filtration, 238 11.7.1 11.7.2 Filter Selection, 238 Disposable Filters, 239 Protocol PI 1.8 Sterile Filtration with Syringe-Tip Filter, 239 Choice of Serum-Free Media, 209 Preparation of Serum-Free Media, 213 Adaptation to Serum-Free Media, 213 Xeno-Free Media, 213 Animal Product-Free Media, 214 Conclusions: Serum-Free Media, 214 Preparation and Sterilization of DPBS-A, 233 Serum Replacements, 209 Use of Serum-Free Medium, 209 10.6.1 10.6.2 10.6.3 Balanced Salt Solutions, 233 Protocol PI 1.4 Development of Serum-Free Medium, 201 Serum-Free Media Formulations, 202 Serum-Free Supplements, 203
10.4.1 10.4.2 Monitoring and Maintenance, 232 Media and Other Reagents, 233 11.6.1 Rationale for Serum-Free Medium, 199 10.1.1 10.1.2 10.1.3 10.2 10.3 10.4 11.6 Protocol PI 1.9 Sterile Filtration with Vacuum Filter Unit, 240 11.7.3 11.7.4 11.8 Reusable Filter Assemblies, 242 Filter Testing, 242 Medium Testing, 242 11.8.1 Sterility Testing, 244 11.8.2 Culture Testing, 244 Suppliers, 247 Suppliers, 214 References, 215 References, 247 11. Preparation and Sterilization, 219 11.1 11.2 Terminology: Preparation, 219 Sterilization Methods, 220 11.2.1 11.2.2 11.2.3 11.2.4 11.2.5 11.2.6 11.2.7 11.3 Dry Heat Sterilization, 220 Pressurized Steam Sterilization (Autoclaving), 221 Irradiation, 223 Plasma Sterilization, 223 Chemical Sterilization, 223 Filter Sterilization, 223 Sterility Indicators, 224 PARTII/ Handling Cultures, 249 12. Aseptic Technique, 291 12.1 12.1.1 12.1.2 12.2 Preparation and Sterilization of Glassware, 224 11.3.1 11.3.2 11.3.3 Detergent Selection, 226 Glassware Sterilization, 226 Caps and Closures, 227 Protocol P1Í.2 Preparation and Sterilization of Screw Caps, 221 12.2.5 12.2.6 12.3 Managing Contamination Risk, 251 Maintaining Sterility, 251 Elements of Aseptic Environment, 252 12.2.1 12.2.2 12.2.3 12.2.4 Glassware, 224 Protocol PI í Λ Objectives of Aseptic Technique, 251 Quiet Area, 253 Laminar Airflow, 253 Work Surface, 255 Personal Protective Equipment (PPE), 256 Reagents and Media, 257 Cultures, 257 Sterile Handling, 258 12.3.1 Swabbing, 258
Contents 12.3.2 12.3.3 12.3.4 12.3.5 12.3.6 12.3.7 12.3.8 12.4 Flaming, 258 Capping, 258 Handling Bottles and Flasks, 258 Pouring, 258 Pipetting, 259 Small-Volume Dispensing, 260 Large-Volume Dispensing, 260 Enrichment of Viable Cells, 292 Protocol Pi3.8 13.8 13.9 Aseptic Technique Using Laminar Airflow, 260 Protocol P12.1 Aseptic Technique: Handling Flasks in a BSC, 260 Protocol Pi2.2 Aseptic Technique: Handling Dishes or Plates, 263 12.4.2 Aseptic Technique on the Open Bench, 263 Protocol ΡΊ2.3 Suppliers, 294 References, 294 Working on the Open Bench, 264 14. D0+306.:C 14.1 14.2 12.5.1 Terminology: Cell Line and Subculture, 297 Initiating a Cell Line, 298 14.2.1 14.2.2 14.2.3 14.3 14.4 Incubators, 265 Cleaning CO? Incubators, 266 12.5.2 Boxed Cultures, 267 12.5.3 Gassing Cultures, 267 Suppliers, 267 14.4.3 14.5 Routine Observation, 304 Standardization of Culture Conditions, 306 Use of Antibiotics, 309 Replacing Medium (Feeding), 309 14.5.1 14.5.2 14.5.3 References, 267 Cell Line Names and Identifiers, 298 Culture Age, 299 Cell Line Validation, 299 Choosing a Cell Line, 300 Maintaining a Cell Line, 304 14.4.1 14.4.2 Controlling Equipment Contamination, 265 Protocol PÍ2.4 Criteria for Replacing Medium, 309 Holding Medium, 309 Standard Procedure for Feeding, 310 Protocol Pi4.1 13. Primary Culture, 269 13.1 13.2 13.2.3 Proteases Used in Disaggregation, 270 Other Agents Used in Disaggregation, 272 Common Features of Disaggregation, 274 13.3.3 13.3.4 Handling Human Biopsies, 280 Primary Explantation, 281 Protocol Pi 3.3 Culture of Primary Expiants, 281 Standard Procedure for
Suspension Culture, 320 Subculture of Suspension Serum-Free Subculture, 322 Record Keeping for Cell Lines, 323 Suppliers, 324 References, 325 Enzymatic Disaggregation, 283 13.5.1 Trypsin, 283 Protocol P13.4 Warm Trypsin Disaggregation, 284 13.5.2 Trypsin with Cold Pre-Exposure, 286 Protocol P13.5 13.5.3 15. Cryopraservation and Banking, 327 15.1 Collagenase Disaggregation, 288 Mechanical Disaggregation, 290 Protocol P13.7 Mechanical Disaggregation by Sieving, 291 Principles of Cryopreservation, 327 15.1.1 15.1.2 15.1.3 15.1.4 Cold Trypsin Disaggregation, 287 Other Enzymatic Procedures, 288 Protocol P13.6 13.6 Growth Cycle and Split Ratios, 319 Protocol P14.3 Cells, 321 14.8 14.9 Trypsinization of Adherent Maintaining Suspension Cultures, 320 14.7.1 Isolation of Mouse Embryos, 275 Chick Embryo, 280 Human Biopsy Samples, 280 Protocol PI3.2 13.5 14.6.4 Non-Human Tissue Samples, 274 Mouse Embryo, 275 Protocol РІЗ. 1 Criteria for Subculture, 312 Dissociation Agents, 312 Standard Procedure for Subculture, 315 Protocol P14.2 Cells, 315 14.7 Feeding Adherent Cultures, 310 Subculture (Passaging), 312 14.6.1 14.6.2 14.6.3 Tissue Acquisition and Isolation, 274 13.3.1 13.3.2 13.4 14.6 Rationale for Primary Culture, 269 Initiation of Primary Culture, 270 13.2.1 13.2.2 13.3 Enrichment of Viable Cells, 292 Record Keeping for Primary Culture, 293 Conclusions: Primary Culture, 294 Good Aseptic Technique, 260 12.4.1 12.5 13.7 XI 15.2 Cryoprotectants, 327 Cooling Rate, 328 Storage Temperature, 328 Vitrification, 329 Apparatus for Cryopreservation, 329 15.2.1 Cryovials, 329
XII Contents 15.2.2 Controlled Cooling Devices, 330 15.2.3 Cryofreezers, 331 15.3 Requirements for Cryopreservation, 335 15.3.1 When to Freeze, 335 15.3.2 Freezing Medium, 335 15.3.3 Cell Concentration, 336 15.4 Cryopreservation Procedures, 336 15.4.1 Cryopreservation in Cryovials, 336 Protocol PI5.1 Freezing Cells in Cryovials, 338 15.4.2 Cryopreservation in Other Vessels, 339 15.4.3 Thawing Stored Cryovials, 339 Protocol PI5.2 Thawing Frozen Cryovials, 339 15.4.4 Viability Testing, 341 15.5 Cell Banking Procedures, 341 15.5.1 Rationale for Cell Banking, 341 15.5.2 Principles of Cell Banking, 341 15.5.3 Replacement of Culture Stocks, 342 15.6 Cell Repositories, 342 15.7 Record Keeping for Frozen Stocks, 345 15.8 Transporting Cells, 347 Suppliers, 348 References, 348 PART V Validation and Characterization, 351 16. Microbial Contamination. 353 16.1 16.2 16.3 16.4 Sources of Contamination, 353 16.1.1 Operator Problems, 356 16.1.2 Environmental Problems, 357 16.1.3 Equipment Problems, 358 16.1.4 Reagent Problems, 358 16.1.5 Cell Line Problems, 359 Management of Contamination, 359 Protocol PI 6.1 Disposal of Contaminated Cultures, 360 Visible Microbial Contamination, 361 16.3.1 Testing of Bacteria, Fungi, and Yeasts, 361 16.3.2 Eradication ofBacteria, Fungi, and Yeasts, 363 Protocol PI 6.2 Treatment of Microbial Contamination, 364 Mycoplasma Contamination, 364 16.4.1 Mycoplasma Detection, 366 Protocol PI 6.3 Detection of Mycoplasma by PCR, 367 Protocol PI 6.4 Detection of Mycoplasma Using Hoechst 33258, 370 16.4.2 Mycoplasma Eradication, 372 Protocol PI 6.5 Eradication of
Mycoplasma Contamination, 372 16.5 Viral Contamination, 373 16.5.1 Detection of Viral Contamination, 374 16.5.2 Eradication ofViral Contamination, 374 16.6 Dealing with Persistent Contamination, 376 Suppliers, 376 References, 376 1]. Cell Une Misirlentificatloii and Authentication, 381 17.1 Terminology: Cross-Contamination, Misidentification, and Authentication, 381 17.2 Misidentified Cell Lines, 382 17.2.1 Impact, 382 17.2.2 Causes, 382 17.2.3 Eradication, 384 17.3 Cell Line Authentication, 386 17.3.1 Evolution of Authentication Techniques, 386 17.3.2 Short Tandem Repeat (STR) Profiling, 389 17.3.3 COI DNA Barcoding, 392 Protocol PÍ 7.1 COI Barcoding of Animal Cells, 394 17.3.4 Cytogenetic Analysis, 398 Protocol P17.2 Chromosome Preparation and Giemsa Staining, 399 17.4 Authentication of Challenging Samples, 401 17.4.1 Cell Line Mixtures, 402 17.4.2 Cell Lines with Microsatellite Instability (MSI), 402 17.4.3 Hybrid Cell Lines, 403 17.5 Conclusions: Authentication, 403 Suppliers, 403 References, 403 IS. Cell Line Characterization, 409 18.1 18.2 18.3 Priorities and Essential Characterization, 409 18.1.1 Validation Testing, 410 18.1.2 Morphology, 410 18.1.3 Growth Curve Analysis, 416 18.1.4 Transformation Assays, 416 Genotype-Based Characterization, 416 18.2.1 Sequence Analysis, 417 18.2.2 Cytogenetic Analysis, 418 18.2.3 Epigenetic Analysis, 418 Phenotype-Based Characterization, 419
Contents 18.3.1 18.3.2 18.3.3 18.3.4 18.4 Cell Line-Specific Markers, 419 Tissue- or Lineage-Specific Markers, 419 Transcriptomic Analysis, 420 Behavioral Assays, 422 Cell Imaging, 423 18.4.1 Microscopy, 423 Protocol PI8.1 Using an Inverted Microscope, 423 18.4.2 Photomicrography, 424 Protocol P18.2 Digital Photography on a Microscope, 425 18.4.3 18.4.4 18.5 Live-Cell Imaging, 426 High-Resolution Imaging, 426 Cell Staining, 428 18.5.1 18.5.2 19.6 Staining with Giemsa, 429 Protocol P18.4 Staining with Crystal Violet, 429 Cell Cycle Analysis, 461 Suppliers, 461 References, 461 PARI VI Physical and Genetic Manipulation. 469 20. Cell Cloning and Selection, 461 20.1 20.2 Terminology: Cloning and Selection, 467 Cloning by Limiting Dilution, 468 20.2.1 Preparation of Cultures for Staining, 428 Histological Stains, 428 Protocol PI 8.3 Dilution Cloning in Dishes, 468 Protocol P20.1 20.2.2 20.3 Soft Agar, 473 Protocol P20.2 20.3.2 References, 430 Adherent Clones, 477 Protocol P20.4 Cell Counting hy Hernocytometer, 440 19.1.2 19.1.3 19.1.4 19.2 Automated Cell Counting, 442 Counting Adherent Cells, 446 Cell Weight and Packed Cell Volume (PCV), 446 20.5 20.6 19.2.2 Dye Uptake Assays, 449 20.6.2 20.6.3 20.7 20.7.4 19.4 Cloning Efficiency, 456 19.4.1 Conclusions: Cloning and Selection, 487 Suppliers, 487 References, 487 Clonogenic Assay for Attached Cells, 457 19.5 Selective Media and Inhibitors, 485 Selective Substrates, 486 Selection by Adhesion and Detachment, 486 Selection by Anchorage-Independent Growth, 486 Clonogenic Assays, 456 Protocol P 19.4 19.4.2 19.4.3 20.8 Preparation of
Feeder Layers, 483 Optimization of Clonal Growth, 484 Selective Culture Conditions, 485 20.7.1 20.7.2 20.7.3 Generating a Growth Curve Using Parameters Derived from the Growth Curve, 455 Cloning on Feeder Layers, 482 Protocol P20.7 Multiwell Plates, 452 19.3.3 Preparation of Conditioned Medium, 481 The Growth Curve, 451 Experimental Design, 452 Protocol PI9.3 Cloning Using Conditioned Medium, 481 Protocol P20.6 Cell Proliferation, 450 19.3.1 19.3.2 Replica Plating, 480 Stimulation of Cloning Efficiency, 481 20.6.1 Cell Counting Using Trypan Blue, 448 Isolation of Suspension Clones, 480 Dye Exclusion Assays, 448 Protocol P19.2 Suspension Clones, 479 Protocol P20.5 Cell Viability, 448 19.2.1 19.3 20.4.2 Manual Cell Counting, 438 Protocol PI 9.1 Isolation of Adherent Clones with Cloning Rings, 477 Cell Counting, 437 19.1.1 Cloning in Methocel, 476 Selection of Clones, 477 20.4.1 19. QuantitatiQn and Growth Kinetics, 43] Cloning in Agar, 474 Methylcellulose (Methocel), 476 Protocol P20.3 20.4 Dilution Cloning, 468 Dilution Cloning in Microwell Plates, 472 Cloning in Suspension, 473 20.3.1 18.5.3 Immunocytochemistry, 430 Suppliers, 430 19.1 Colony Counting, 459 Analysis of Colony Formation, 459 DNA Synthesis, 460 XIII 21. Cell Separation and Sorting, 491 21.1 Cell Density and Isopycnic Centrifugation, 491
XIV Contents 21.1.1 22.4.2 22.4.3 Toxicity, 527 Indels and Rearrangements at the Target Site, 527 22.4.4 Off-Target Effects, 527 22.4.5 Oncogenesis, 528 Suppliers, 528 Density Gradient Centrifugation, 491 Protocol P2Í.Í Cell Separation by Centrifugation on a Density Gradient, 493 21.1.2 21.2 Cell Size and Sedimentation Velocity, 495 21.2.1 21.2.2 21.3 Optimization of Density Gradients, 495 Velocity Sedimentation at Unit Gravity, 495 Centrifugal Elutriation, 496 Magnetic Separation and Sorting, 496 Protocol P2Í.2 Magnet-Activated Cell Sorting (MACS), 499 21.4 21.5 Fluorescence-Activated Cell Sorting (FACS), 500 Microfluidic Sorting, 502 Minireview М2 І A 21.6 Microfluidic Cell Culture, 503 Conclusions: Sorting and Separation, 505 Suppliers, 505 PARI VII Stem Cells and Differentiated Cells, 035 23. Culture of Stem Cells, 537 23.1 23.2 References, 505 ......................................................... 23.3 Gene Delivery, 509 22.1.1 23.4 Transfection with Calcium Phosphate, 511 22.1.3 22.1.4 22.2 22.2.1 22.2.2 22.2.3 Zinc Finger Nucleases (ZFNs), 517 Transcription Activator-Like Effector Nucleases (TALENs), 517 CRISPR/Cas RNA-Guided Nucleases, 519 Cryopreservation and Thawing, 554 23.5 23.6 23.7 23.8 Perinatal Stem Cells, 556 Adult Stem Cells, 557 Stem Cell Characterization and Banking, 558 Conclusions: Culture of Stem Cells, 560 Suppliers, 561 References, 561 Immortalization, 523 22.3.1 22.3.2 22.3.3 Early Immortalization Strategies, 523 Immortalization Using Viral Genes and Oncogenes, 523 Immortalization Using Telomerase, 524 Protocol P22.4 Immortalization Using ItTERT
Transfection, 524 22.3.4 22.4 Evolution in Culture ofhPSCs, 549 Culture Conditions, 550 Feeding and Subculture, 551 Protocol P23.3 Cryopreservation Using ROCK Inhibitor, 555 Electroporation, 514 Viral Transduction, 514 Protocol P22.3 Delivery of CRISPR/Cas9 RNP Using Electroporation, 522 22.3 23.4.4 Optimization of Lipofection, 513 Gene Editing, 517 Human Pluripotent Stem Cell (hPSC) Lines, 549 Protocol P23.2 Subculture in Chemically Defined Conditions, 551 Transfection with Cationic Lipids and Polymers, 512 Protocol P22.2 Induction of Pluripotency, 545 23.4.1 23.4.2 23.4.3 Protocol P22.1 Calcium Phosphate Coprecipitation, 511 22.1.2 Mouse (mESCs), 540 Human (hESCs), 542 Other Species, 545 Protocol P23.1 Generation of iPSCs Using Sendai Viral Vectors, 547 Immortalization, 509 22.1 Terminology: Stem Cells, 537 Embryonic Stem Cells (ESCs), 540 23.2.1 23.2.2 23.2.3 22 5^4012011^7084 .“ References, 528 Conditional Reprogramming, 525 Screening and Artifacts, 526 22.4.1 Selection of Modified Cells, 526 24. Culture uf Specific Cell Types, 507 24.1 24.2 Specialized Cells and Their Availability, 567 Epithelial Cells, 572 24.2.1 24.2.2 24.2.3 24.2.4 Epidermis, 572 Cornea, 573 Oral Epithelium, 573 Bronchial and Tracheal Epithelium, 574
Contents 24.2.5 Gastrointestinal Tract, 574 24.2.6 Liver, 574 24.2.7 Pancreas, 574 24.2.8 Breast, 575 24.2.9 Cervix, 577 24.2.10 Prostate, 577 Mesenchymal Cells, 577 24.3 24.3.1 Connective Tissue, 577 24.3.2 Adipose Tissue, 578 24.3.3 Muscle, 579 24.3.4 Cartilage, 579 24.3.5 Bone, 579 24.3.6 Endothelium, 580 24.4 Neuroectodermal Cells, 580 24.4.1 Neurons, 580 24.4.2 Glial Cells, 580 24.4.3 Endocrine Cells, 581 24.4.4 Melanocytes, 581 24.5 Hematopoietic Cells, 581 24.5.1 Lymphoid Cells, 581 24.5.2 Macrophages and Myeloid Cells, 582 24.5.3 Erythroid Cells, 583 24.5.4 Hybridoma Cells, 583 24.5.5 Chimeric Antigen Receptor (CAR) Т-Cells (CAR T-Cells), 584 24.6 Culture of Cells from Poikilotherms, 585 24.6.1 Fish Cells, 586 24.6.2 Insect Cells, 586 Suppliers, 587 References, 587 25. Culture of Tumor Cells, 593 25.1 25.2 25.3 25.4 Challenges of Tumor Cell Culture, 593 Primary Culture of Tumor Cells, 594 25.2.1 Selection of Representative Cells, 594 Protocol P25.1 Freezing Tumor Biopsies, 594 25.2.2 Disaggregation of Tumor Samples, 596 Development of Tumor Cell Lines, 596 25.3.1 Subculture of Primary Tumor Cultures, 596 25.3.2 Continuous Tumor Cell Lines, 596 25.3.3 Validation ofTumor Cell Lines, 598 25.3.4 Characterization ofTumor Cell Lines, 598 Selective Culture of Tumor Cells, 599 25.4.1 Selective Media and Techniques, 599 25.4.2 Confluent Feeder Layers, 599 Protocol P25.2 Growth on Confluent Feeder Layers, 600 25.4.3 Suspension Cloning, 601 25.4.4 Spheroid Culture, 603 25.4.5 Xenografts, 603 XV 25.5 Specific Tumor Types, 603 25.5.1 Carcinoma, 604 25.5.2 Sarcoma, 606 25.5.3
Melanoma, 606 25.5.4 Lymphoma and Leukemia, 606 25.5.5 Glioma, 606 25.6 Cancer Stem Cells (CSCs), 606 Minireview M25.1 Culture of Cancer Stem Cells, 606 Suppliers, 608 References, 608 26. Differentiation, 616 In Vitro Models of Differentiation, 615 Differentiation Status in Culture, 617 26.2.1 Differentiation and Malignancy, 617 26.2.2 Proliferation and Differentiation, 618 26.2.3 Dedifferentiation, 618 26.2.4 Transdifferentiation, 618 26.2.5 Epithelial-Mesenchymal Transition (EMT), 619 26.3 Induction of Differentiation, 620 26.3.1 Exogenous Soluble Factors, 620 26.3.2 Endogenous Factors, 622 26.3.3 Geometry and Polarity, 625 26.3.4 Cell-Cell Interactions, 625 26.3.5 Cell-Extracellular Matrix (ECM) Interactions, 626 26.3.6 Air-Liquid Interface, 628 26.3.7 Biomechanical Regulation, 628 26.4 Practical Aspects, 628 26.5 Ongoing Challenges, 629 26.5.1 Markers of Differentiation, 629 26.5.2 Stem Cell Differentiation, 629 Suppliers, 631 References, 631 26.1 26.2 PARI VIII Model Environmentsand Applications, 639 11 Three-Dimensional Coltore, 641 . 27.1 27.2 27.3 Terminology: 3D Culture, 641 Technologies for 3D Culture, 643 Minireview М27.1 Advances in Technologies Enabling 3D Cell Culture and the Formation of Tissue-Like Architecture In Vitro, 643 Benefits and Limitations of 3D Culture, 646
XVI Contents 27.4 Scaffold-Free 3D Culture Systems, 647 27.4.1 Spheroid Culture, 647 Protocol P27.1 Tumor Spheroid Formation and Embedding, 649 27.4.2 Dynamic Culture Systems, 650 27.5 Scaffold-Based 3D Culture Systems, 652 27.5.1 Overlay, Embedding, and Encapsulation, 652 27.5.2 Filter Well Inserts, 655 Protocol P27.2 Culture Using Filter Well Inserts, 65 7 27.5.3 Hollow Fiber Systems, 658 27.5.4 Microcarriers and Macrocarriers, 658 27.6 Organoid Culture, 659 27.7 Organotypic Culture, 660 27.7.1 Tissue Equivalents, 660 27.7.2 Tissue Engineering, 660 27.8 Organ Culture, 662 27.9 Characterization of 3D Cultures, 662 Suppliers, 663 References, 663 28. Scale-Up anti Automation, 669 28.1 Terminology: Scale-Up and Bioreactors, 669 28.2 Scale-Up in Suspension, 671 28.2.1 Spinner Culture, 671 28.2.2 Single-Use Bioreactor Systems, 673 28.2.3 Scaffold-Free Perfusion Bioreactors, 675 28.2.4 Other Bioreactor Systems for Suspension Culture, 675 28.3 Scale-Up in Monolayer, 677 28.3.1 Roller Culture, 677 28.3.2 Multisurface Propagators, 678 Protocol P28.1 Handling a Nunc Cell Factory, 678 28.3.3 Microcarrier Culture, 681 28.3.4 Scaffold-Based Perfusion Bioreactors, 684 28.4 Monitoring and Process Control, 685 28.5 Scale-Up for Manufacture, 688 Minireview M28. Í Culture Scale-Up and Bioreactors, 688 28.6 High-Throughput Screening, 691 28.7 Automation and Bioprinting, 691 28.7.1 Automation of Culture Handling, 692 28.7.2 Automation of Cell-Based Assays, 692 28.7.3 3D Bioprinting, 694 Suppliers, 696 References, 696 29. Toxicity Testing, 701 29.1 In Vitro Toxicity Testing, 701 29.1.1
Applications, 702 29.1.2 Limitations, 703 29.1.3 Requirements, 703 29.2 Cytotoxicity Assays, 704 29.2.1 Selecting a Cytotoxicity Assay, 704 29.2.2 Assays Based on Cell Metabolism, 704 Protocol P29. Í MTT-Based Cytotoxicity Assay, 707 29.2.3 Assays Based on Cell Death, 709 29.2.4 Assays Based on Cell Survival, 710 29.2.5 Analysis of Cytotoxicity Assays, 711 29.3 Genotoxicity Assays, 715 29.4 Carcinogenicity Assays, 716 29.5 Advanced Models for Toxicity Testing, 716 29.5.1 3D Models for Eye and Skin Irritation, 716 29.5.2 Organ-on-Chip Technologies, 717 Suppliers, 719 References, 719 PART IX Teaching and Troubleshooting, 728 30. Training, 727 30.1 Training Principles, 727 30.1.1 Roles and Responsibilities, 727 30.1.2 Induction, 728 30.1.3 Training Documents, 729 30.1.4 Hands-on Training, 729 30.2 Training Programs, 729 30.2.1 Topics, 729 30.2.2 Exercises, 731 References, 731 31. Problem Solving, 733 31.1 31.2 31.3 31.4 Microbial Contamination, 733 31.1.1 Type of Microbial Contamination, 734 31.1.2 Contamination Is Limited to One Person, 734 31.1.3 Contamination Is Widespread, 736 31.1.4 Problems with Laminar Flow or Air Quality, 737 Cross-Contamination and Misidentification, 737 Chemical Contamination, 738 Slow Cell Growth, 738
Contents 31.4.1 Problem Is Limited to One Person, 739 31.4.2 Problem Is Widespread, 739 31.5 Abnormal Cell Appearance, 740 31.6 Problems with Materials, 741 31.6.1 Culture Vessels, 741 31.6.2 Medium Formulation and Preparation, 741 31.6.3 Medium Stability and Storage, 743 31.6.4 Water Quality, 743 31.7 Problems with Primary Culture, 744 31.7.1 Suspected Contamination, 744 31.7.2 Poor Take in Primary Culture, 744 31.7.3 Incorrect Phenotype after Primary Culture, 745 31.8 Problems with Feeding or Subculture, 746 31.8.1 pH after Feeding, 746 31.8.2 Poor Take after Subculture, 746 31.8.3 Uneven Growth after Subculture, 747 31.9 Problems with Cryopreservation, 748 31.9.1 Loss of Frozen Stocks, 748 31.9.2 Poor Viability after Thawing, 749 31.9.3 Changed Appearance after Thawing, 750 31.10 Problems with Cloning, 750 31.10.1 Too Few Colonies per Dish, 750 31.10.2 Too Many Colonies per Dish, 751 31.10.3 Non-Random Distribution of Colonies, 751 31.10.4 Incubation of Cloning Dishes, 752 References, 752 32. In Conclusion, 753 Appendix A Appendix В Glossary, 755 Calculations and Preparation of Reagents, 761 Calculations, 761 Counting Cells with a Hemacytometer, 761 Dilution of a Cell Suspension, 761 Population Doubling Level (PDL), 761 Molarity, 762 Percentages and Dilutions, 762 Presane, 762 kotor Speed (rpm to g), 762 XVII Preparation of Reagents, 762 Acetic Acid: Methanol, 762 Agar (2.5%), 762 Alcohol (70%), 762 Bacto™ Peptone (5%), 763 Balanced Salt Solutions, 763 Carboxymethylcellulose (CMC; 4%), 763 Chick Embryo Extract, 763 Collagénase, 763 Collection Medium, 763 Crystal Violet
(0.1%), 764 Dexamethasone (1 mg/ml), 764 Dissection Balanced Salt Solution (DBSS), 764 Dulbecco’s Phosphate-Buffered Saline Without Ca2+ and Mg2+ (DPBS-A), 764 ED7A (10 mM in DPBS-A), 764 EGTA, 764 Erythrosin В, 764 Gelatin (1%), 765 Giemsa Stain, 765 Glucose (20%), 765 Glutamine, 200 mM, 765 Hanks’s Balanced Salt Solution (HBSS), 765 HAT Medium, 765 HB Medium, 765 HEPES, 765 Hoechst 33258, 766 Media, 766 2-Mcrcaptocthanol (ß-Mercaptoethanol; 0.1 M), 766 Methylcellulose (Methocel, 1.6%), 766 Mitomycin C (100 pg/ml), 766 MTT (50 mg/ml), 766 N2 Supplement, 766 N2B27 Medium, 767 Naphthalene Black (Amido Black; 1%), 767 Non-essential Amino Acids (NEAA, lOOx), 767 Paraformaldehyde (4%), 767 Trypan Blue (0.4%), 767 Trypsin (2.5%), 768 Versene, 768 Suppliers, 768 References, 768 Appendix C Media Formulations, 769 References, 779 Index, 781
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Foreword, xix Acknowledgments, xxi Abbreviations, xxiii Book Navigation, xxix PART I Understanding Cel! Culture, 1 1. Introduction, 3 1.1 Terminology, 3 1.1.1 Tissue Culture and Cell Culture, 3 1.1.2 Sources of Terminology, 3 1.2 Historical Development, 4 1.2.1 Substrates and Media, 4 1.2.2 Primary Cultures and Cell Lines, 5 1.2.3 Organ, Organotypic, and Organoid Culture, 7 1.3 Applications, 12 1.4 Advantages of Tissue Culture, 13 1.4.1 Environmental Control, 13 1.4.2 Homogeneity and Characterization, 14 1.4.3 Economy, Scale, and Automation, 14 1.4.4 Replacement of In Vivo Models, 14 1.5 Limitations of Tissue Culture, 15 1.5.1 Quality and Expertise, 15 1.5.2 Quantity and Cost, 16 1.5.3 Limited Species and Cell Types, 17 1.5.4 Limited Understanding of the Cell and its Microenvironment, 17 References, 18 l. Biology of Cultured Culls, 33 2.1 2.2 The Culture Environment, 23 Cell Adhesion, 23 2.2.1 Intercellular Junctions, 25 2.2.2 Cell Adhesion Molecules, 25 2.2.3 Cytoskeleton, 26 2.2.4 Extracellular Matrix (ECM), 27 2.2.5 Cell Motility, 27 2.3 Cell Division, 28 2.3.1 Cell Cycle, 28 2.3.2 Control of the Cell Cycle, 29 2.4 Cell Fate, 30 2.4.1 Embryonic Lineages, 30 2.4.2 Stem Cells and Potency, 31 2.4.3 Differentiation, 31 2.4.4 Control of Potency and Differentiation, 32 2.4.5 Lineage Commitment, 33 2.4.6 Lineage Plasticity, 35 2.5 Cell Death, 35 References, 36 3. Origin and Mtion of Cultured Cells, 39 3.1 3.2 Origin of Cultured Cells, 39 3.1.1 Sample Origin, 39 3.1.2 Disease Origin, 40 Evolution of Cell Lines, 40 3.2.1 Phases of Cell Cultivation, 40 3.2.2 Clonal Evolution, 41
VII
VIII Contents 3.3 Changes in Genotype, 43 3.3.1 3.3.2 3.4 Phenotypic Variation, 46 Phenotype and Culture Conditions, 47 5.3 Senescence and Immortalization, 48 Minireview М3.1 Senescence and 3.6 Intrinsic Control of Senescence, 50 Extrinsic Control of Senescence, 50 5.4 3.6.1 3.6.2 3.6.3 Characteristics of Transformation, 50 Aberrant Growth Control, 52 Tumorigenicity and Malignancy, 55 5.5 3.7 Conclusions: Origin and Evolution, 58 References, 58 РАШИ ^908298^334812 " ՛ . 'V. Requirements, 63 4. laboratory Design and Layout, 66 4.1 4.2 Sterile Handling Area, 75 Incubation Area, 76 Quarantine Area, 77 Preparation Area, 77 Washup Area, 77 Storage Area, 78 6. Safety and Bioethics, 111 6.1 6.2 6.3 5.1.4 6.4 Needlestick and Sharps Injuries, 118 Hazardous Substances, 118 Asphyxia and Explosion, 119 Burns and Frostbite, 120 Fire, 120 Equipment Hazards, 120 Biosafety, 121 6.3.6 6.3.7 6. Equipment and Materials, 86 Biological Safety Cabinet (BSC), 85 BSC Services and Consumables, 88 Sterile Liquid Handling Equipment, 90 Centrifuge, 94 Hazards in Tissue Culture Laboratories, 117 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 References, 83 5.1.1 5.1.2 5.1.3 Risk Assessment, 111 Safety Regulations, 113 Training, 116 Ergonomics, 117 6.2.1 6.2.2 6.2.3 6.2.4 6.2.5 6.2.6 Contingency Plans and Priorities, 81 Equipment Monitoring and Alarms, 83 5.1 Sterile Handling Area Equipment, 85 Laboratory Safety, 111 6.1.1 6.1.2 6.1.3 6.1.4 Disaster and Contingency Planning, 80 4.3.1 4.3.2 Cold Storage Equipment, 107 References, 109 Layout of Laboratory Areas, 74 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.3 General Design
Considerations, 65 User Requirements, 67 Regulatory Requirements, 70 Engineering Requirements, 71 Water Purification Systems, 104 Preparation Equipment, 104 Washup Equipment, 105 Sterilization Equipment, 106 5.5.1 Refrigerators and Freezers, 107 5.5.2 Cryofreezers, 108 5.5.3 Rate-Controlled Freezer, 108 Suppliers, 108 Design Requirements, 65 4.1.1 4.1.2 4.1.3 4.1.4 Incubators, 100 Incubator Accessories, 103 Water Baths, 103 Preparation and Washup Equipment, 104 5.4.1 5.4.2 5.4.3 5.4.4 Transformation, 50 Microscopes, 97 Cameras, 98 Computer and Monitor, 98 Cell Counting and Analysis Equipment, 99 Incubation Equipment, 99 5.3.1 5.3.2 5.3.3 Immortalization, 48 3.5.1 3.5.2 Imaging and Analysis Equipment, 97 5.2.1 5.2.2 5.2.3 5.2.4 Changes in Phenotype, 46 3.4.1 3.4.2 3.5 5.2 Chromosomal Aberrations, 43 Genomic Variation, 44 Source of Biohazard Risk, 121 Biohazard Risk Groups, 122 Biological Containment Levels, 123 Containment Equipment, 123 Decontamination and Fumigation, 128 Waste Disposal and Disinfectants, 128 Genetically Modified Organisms (GMOs), 129 Bioethics, 129 6.4.1 Ethical Use of Animal Tissue,130 6.4.2 Ethical Use of Human Tissue,130 6.4.3 Donor Consent, 131 Suppliers, 132 References, 132
Contents 8.3.3 8.3.4 8.3.5 8.3.6 7. Reproducibility and Good Cell Colture Practice, 137 7.1 Reproducibility, 137 7.1.1 7.1.2 7.1.3 7.2 7.2.2 7.2.3 7.4.2 7.5.2 7.5.3 7.5.4 7.5.5 References, 173 Standard Operating Procedures (SOPs), 148 Media and Reagents, 149 Culture Vessels, 149 Equipment, 149 Facilities, 149 9. Defined Media and Supplements, 177 9.1 9.2 pH, 177 Buffering, 178 Carbon Dioxide (C02) and Sodium Bicarbonate (NaHC03), 178 9.2.4 Oxygen, 179 Minireview M9A Hypoxic Cell Culture, 180 9.2.5 Temperature, 183 9.2.6 Osmolality, 184 9.2.7 Viscosity, 185 9.2.8 Surface Tension and Foaming, 185 Replicate Sampling, 150 References, 151 ^ Requirements, 155 9.3 9.4 8.2 8.2.1 8.2.2 8.3 9.5 Substrate Treatments, 159 8.3.1 8.3.2 9.6 Amino Acids, 188 Vitamins, 188 Inorganic Salts, 188 Glucose, 188 Other Components, 188 Serum, 189 9.5.1 9.5.2 9.5.3 9.5.4 9.5.5 9.5.6 Common Substrate Materials, 158 Alternative Substrate Materials, 158 Substrate Conditioning, 160 Extracellular Matrix (ECM) Coatings, 160 Protocol P8. Í Application of Matrigel Coatings, 1 60 Balanced Salt Solutions, 185 Media Formulations, 186 9.4.1 9.4.2 9.4.3 9.4.4 9.4.5 R. Culture Vessels and Substrates, 157 Attachment and Growth Requirements, 157 Substrate Materials, 158 Medium Development, 177 Physicochemical Properties, 177 9.2.1 9.2.2 9.2.3 7.6.1 Experimental Design, 150 7.6.2 Samples and Data, 150 Suppliers, 151 8.1 Application-Specific Vessels, 170 8.6.1 Imaging, 170 8.6.2 Suspension Culture, 171 8.6.3 Scaffold-Free 3D Culture, 171 8.6.4 Permeable Supports, 172 8.6.5 Scaffold-Based 3D Culture, 172 Suppliers,
172 Provenance Information, 145 Reporting for Publication, 146 Testing for Microbial Contamination, 147 Testing for Authenticity, 147 Cell Yield, 166 Multiwell Plates, 166 Flasks and Petri Dishes, 166 Multilayer Flasks, 166 Lids and Venting, 167 Uneven Growth, 169 Cost, 169 Quality Assurance (QA), 148 7.5.1 7.6 8.6 Validation Testing, 146 7.4.1 7.5 Good Cell Culture Practice (GCCP), 142 Good Laboratory Practice (GLP), 142 Good Manufacturing Practice (GMP), 143 Feeder Layers, 163 Choice of Culture Vessel, 164 8.5.1 8.5.2 8.5.3 8.5.4 8.5.5 8.5.6 8.5.7 Cell Line Provenance, 145 7.3.1 7.3.2 7.4 8.3.7 8.4 8.5 Good Practice Requirements, 141 7.2.1 7.3 Terminology: Reproducible Research, 137 Causes of Irreproducible Research, 138 Solutions to Irreproducible Research, 139 Collagen and Gelatin, 161 ECM Mimetic Treatments, 161 Polymer Coatings, 161 Non-Adhesive Substrates and Patterning, 163 Other Surface Treatments, 163 Protein, 189 Hormones and Growth Factors, 190 Nutrients and Metabolites, 190 Lipids, 190 Trace Elements, 190 Inhibitors, 191 Other Media Supplements, 191 9.6.1 9.6.2 Conditioned Medium, 191 Antibiotics, 191 IX
x Contents 9.7 Choice of Complete Medium, 191 9.7.1 9.7.2 9.7.3 9.8 Serum Testing, 193 Serum Batch Reservation, 193 Serum Treatment, 193 11.4 Other Laboratory Apparatus, 229 11.4.1 11.4.2 11.5 Water, 229 11.5.1 11.5.2 Storage of Medium and Serum, 194 Suppliers, 194 References, 194 Cleaning and Packaging, 229 Sterilization or Disinfection, 229 Water Purity, 229 Purification Methods, 230 Protocol P1Í.3 Preparation and Sterilization of Ultrapure Water (UPW), 230 11.5.3 10. Serum-Free Media, 199 10.1 10.4.3 10.5 10.6 Disadvantages of Serum, 199 Advantages of Serum-Free Media, 200 Disadvantages of Serum-Free Media, 201 Hormones and Growth Factors, 203 Antioxidants, Vitamins, and Lipids, 208 Other Supplements for Serum-Free Medium, 208 10.7 10.8 10.9 11.6.2 Basal and Complete Media, 234 Protocol PI 1.5 Preparation of Medium from Powder, 235 Protocol PI 1.6 Preparation of Medium from Юх Concentrate, 236 Protocol PI 1.7 Preparation of Medium from lx Stock, 237 11.6.3 11.7 Serum, 238 Sterile Filtration, 238 11.7.1 11.7.2 Filter Selection, 238 Disposable Filters, 239 Protocol PI 1.8 Sterile Filtration with Syringe-Tip Filter, 239 Choice of Serum-Free Media, 209 Preparation of Serum-Free Media, 213 Adaptation to Serum-Free Media, 213 Xeno-Free Media, 213 Animal Product-Free Media, 214 Conclusions: Serum-Free Media, 214 Preparation and Sterilization of DPBS-A, 233 Serum Replacements, 209 Use of Serum-Free Medium, 209 10.6.1 10.6.2 10.6.3 Balanced Salt Solutions, 233 Protocol PI 1.4 Development of Serum-Free Medium, 201 Serum-Free Media Formulations, 202 Serum-Free Supplements, 203
10.4.1 10.4.2 Monitoring and Maintenance, 232 Media and Other Reagents, 233 11.6.1 Rationale for Serum-Free Medium, 199 10.1.1 10.1.2 10.1.3 10.2 10.3 10.4 11.6 Protocol PI 1.9 Sterile Filtration with Vacuum Filter Unit, 240 11.7.3 11.7.4 11.8 Reusable Filter Assemblies, 242 Filter Testing, 242 Medium Testing, 242 11.8.1 Sterility Testing, 244 11.8.2 Culture Testing, 244 Suppliers, 247 Suppliers, 214 References, 215 References, 247 11. Preparation and Sterilization, 219 11.1 11.2 Terminology: Preparation, 219 Sterilization Methods, 220 11.2.1 11.2.2 11.2.3 11.2.4 11.2.5 11.2.6 11.2.7 11.3 Dry Heat Sterilization, 220 Pressurized Steam Sterilization (Autoclaving), 221 Irradiation, 223 Plasma Sterilization, 223 Chemical Sterilization, 223 Filter Sterilization, 223 Sterility Indicators, 224 PARTII/ Handling Cultures, 249 12. Aseptic Technique, 291 12.1 12.1.1 12.1.2 12.2 Preparation and Sterilization of Glassware, 224 11.3.1 11.3.2 11.3.3 Detergent Selection, 226 Glassware Sterilization, 226 Caps and Closures, 227 Protocol P1Í.2 Preparation and Sterilization of Screw Caps, 221 12.2.5 12.2.6 12.3 Managing Contamination Risk, 251 Maintaining Sterility, 251 Elements of Aseptic Environment, 252 12.2.1 12.2.2 12.2.3 12.2.4 Glassware, 224 Protocol PI í Λ Objectives of Aseptic Technique, 251 Quiet Area, 253 Laminar Airflow, 253 Work Surface, 255 Personal Protective Equipment (PPE), 256 Reagents and Media, 257 Cultures, 257 Sterile Handling, 258 12.3.1 Swabbing, 258
Contents 12.3.2 12.3.3 12.3.4 12.3.5 12.3.6 12.3.7 12.3.8 12.4 Flaming, 258 Capping, 258 Handling Bottles and Flasks, 258 Pouring, 258 Pipetting, 259 Small-Volume Dispensing, 260 Large-Volume Dispensing, 260 Enrichment of Viable Cells, 292 Protocol Pi3.8 13.8 13.9 Aseptic Technique Using Laminar Airflow, 260 Protocol P12.1 Aseptic Technique: Handling Flasks in a BSC, 260 Protocol Pi2.2 Aseptic Technique: Handling Dishes or Plates, 263 12.4.2 Aseptic Technique on the Open Bench, 263 Protocol ΡΊ2.3 Suppliers, 294 References, 294 Working on the Open Bench, 264 14. D0+306.:C 14.1 14.2 12.5.1 Terminology: Cell Line and Subculture, 297 Initiating a Cell Line, 298 14.2.1 14.2.2 14.2.3 14.3 14.4 Incubators, 265 Cleaning CO? Incubators, 266 12.5.2 Boxed Cultures, 267 12.5.3 Gassing Cultures, 267 Suppliers, 267 14.4.3 14.5 Routine Observation, 304 Standardization of Culture Conditions, 306 Use of Antibiotics, 309 Replacing Medium (Feeding), 309 14.5.1 14.5.2 14.5.3 References, 267 Cell Line Names and Identifiers, 298 Culture Age, 299 Cell Line Validation, 299 Choosing a Cell Line, 300 Maintaining a Cell Line, 304 14.4.1 14.4.2 Controlling Equipment Contamination, 265 Protocol PÍ2.4 Criteria for Replacing Medium, 309 Holding Medium, 309 Standard Procedure for Feeding, 310 Protocol Pi4.1 13. Primary Culture, 269 13.1 13.2 13.2.3 Proteases Used in Disaggregation, 270 Other Agents Used in Disaggregation, 272 Common Features of Disaggregation, 274 13.3.3 13.3.4 Handling Human Biopsies, 280 Primary Explantation, 281 Protocol Pi 3.3 Culture of Primary Expiants, 281 Standard Procedure for
Suspension Culture, 320 Subculture of Suspension Serum-Free Subculture, 322 Record Keeping for Cell Lines, 323 Suppliers, 324 References, 325 Enzymatic Disaggregation, 283 13.5.1 Trypsin, 283 Protocol P13.4 Warm Trypsin Disaggregation, 284 13.5.2 Trypsin with Cold Pre-Exposure, 286 Protocol P13.5 13.5.3 15. Cryopraservation and Banking, 327 15.1 Collagenase Disaggregation, 288 Mechanical Disaggregation, 290 Protocol P13.7 Mechanical Disaggregation by Sieving, 291 Principles of Cryopreservation, 327 15.1.1 15.1.2 15.1.3 15.1.4 Cold Trypsin Disaggregation, 287 Other Enzymatic Procedures, 288 Protocol P13.6 13.6 Growth Cycle and Split Ratios, 319 Protocol P14.3 Cells, 321 14.8 14.9 Trypsinization of Adherent Maintaining Suspension Cultures, 320 14.7.1 Isolation of Mouse Embryos, 275 Chick Embryo, 280 Human Biopsy Samples, 280 Protocol PI3.2 13.5 14.6.4 Non-Human Tissue Samples, 274 Mouse Embryo, 275 Protocol РІЗ. 1 Criteria for Subculture, 312 Dissociation Agents, 312 Standard Procedure for Subculture, 315 Protocol P14.2 Cells, 315 14.7 Feeding Adherent Cultures, 310 Subculture (Passaging), 312 14.6.1 14.6.2 14.6.3 Tissue Acquisition and Isolation, 274 13.3.1 13.3.2 13.4 14.6 Rationale for Primary Culture, 269 Initiation of Primary Culture, 270 13.2.1 13.2.2 13.3 Enrichment of Viable Cells, 292 Record Keeping for Primary Culture, 293 Conclusions: Primary Culture, 294 Good Aseptic Technique, 260 12.4.1 12.5 13.7 XI 15.2 Cryoprotectants, 327 Cooling Rate, 328 Storage Temperature, 328 Vitrification, 329 Apparatus for Cryopreservation, 329 15.2.1 Cryovials, 329
XII Contents 15.2.2 Controlled Cooling Devices, 330 15.2.3 Cryofreezers, 331 15.3 Requirements for Cryopreservation, 335 15.3.1 When to Freeze, 335 15.3.2 Freezing Medium, 335 15.3.3 Cell Concentration, 336 15.4 Cryopreservation Procedures, 336 15.4.1 Cryopreservation in Cryovials, 336 Protocol PI5.1 Freezing Cells in Cryovials, 338 15.4.2 Cryopreservation in Other Vessels, 339 15.4.3 Thawing Stored Cryovials, 339 Protocol PI5.2 Thawing Frozen Cryovials, 339 15.4.4 Viability Testing, 341 15.5 Cell Banking Procedures, 341 15.5.1 Rationale for Cell Banking, 341 15.5.2 Principles of Cell Banking, 341 15.5.3 Replacement of Culture Stocks, 342 15.6 Cell Repositories, 342 15.7 Record Keeping for Frozen Stocks, 345 15.8 Transporting Cells, 347 Suppliers, 348 References, 348 PART V Validation and Characterization, 351 16. Microbial Contamination. 353 16.1 16.2 16.3 16.4 Sources of Contamination, 353 16.1.1 Operator Problems, 356 16.1.2 Environmental Problems, 357 16.1.3 Equipment Problems, 358 16.1.4 Reagent Problems, 358 16.1.5 Cell Line Problems, 359 Management of Contamination, 359 Protocol PI 6.1 Disposal of Contaminated Cultures, 360 Visible Microbial Contamination, 361 16.3.1 Testing of Bacteria, Fungi, and Yeasts, 361 16.3.2 Eradication ofBacteria, Fungi, and Yeasts, 363 Protocol PI 6.2 Treatment of Microbial Contamination, 364 Mycoplasma Contamination, 364 16.4.1 Mycoplasma Detection, 366 Protocol PI 6.3 Detection of Mycoplasma by PCR, 367 Protocol PI 6.4 Detection of Mycoplasma Using Hoechst 33258, 370 16.4.2 Mycoplasma Eradication, 372 Protocol PI 6.5 Eradication of
Mycoplasma Contamination, 372 16.5 Viral Contamination, 373 16.5.1 Detection of Viral Contamination, 374 16.5.2 Eradication ofViral Contamination, 374 16.6 Dealing with Persistent Contamination, 376 Suppliers, 376 References, 376 1]. Cell Une Misirlentificatloii and Authentication, 381 17.1 Terminology: Cross-Contamination, Misidentification, and Authentication, 381 17.2 Misidentified Cell Lines, 382 17.2.1 Impact, 382 17.2.2 Causes, 382 17.2.3 Eradication, 384 17.3 Cell Line Authentication, 386 17.3.1 Evolution of Authentication Techniques, 386 17.3.2 Short Tandem Repeat (STR) Profiling, 389 17.3.3 COI DNA Barcoding, 392 Protocol PÍ 7.1 COI Barcoding of Animal Cells, 394 17.3.4 Cytogenetic Analysis, 398 Protocol P17.2 Chromosome Preparation and Giemsa Staining, 399 17.4 Authentication of Challenging Samples, 401 17.4.1 Cell Line Mixtures, 402 17.4.2 Cell Lines with Microsatellite Instability (MSI), 402 17.4.3 Hybrid Cell Lines, 403 17.5 Conclusions: Authentication, 403 Suppliers, 403 References, 403 IS. Cell Line Characterization, 409 18.1 18.2 18.3 Priorities and Essential Characterization, 409 18.1.1 Validation Testing, 410 18.1.2 Morphology, 410 18.1.3 Growth Curve Analysis, 416 18.1.4 Transformation Assays, 416 Genotype-Based Characterization, 416 18.2.1 Sequence Analysis, 417 18.2.2 Cytogenetic Analysis, 418 18.2.3 Epigenetic Analysis, 418 Phenotype-Based Characterization, 419
Contents 18.3.1 18.3.2 18.3.3 18.3.4 18.4 Cell Line-Specific Markers, 419 Tissue- or Lineage-Specific Markers, 419 Transcriptomic Analysis, 420 Behavioral Assays, 422 Cell Imaging, 423 18.4.1 Microscopy, 423 Protocol PI8.1 Using an Inverted Microscope, 423 18.4.2 Photomicrography, 424 Protocol P18.2 Digital Photography on a Microscope, 425 18.4.3 18.4.4 18.5 Live-Cell Imaging, 426 High-Resolution Imaging, 426 Cell Staining, 428 18.5.1 18.5.2 19.6 Staining with Giemsa, 429 Protocol P18.4 Staining with Crystal Violet, 429 Cell Cycle Analysis, 461 Suppliers, 461 References, 461 PARI VI Physical and Genetic Manipulation. 469 20. Cell Cloning and Selection, 461 20.1 20.2 Terminology: Cloning and Selection, 467 Cloning by Limiting Dilution, 468 20.2.1 Preparation of Cultures for Staining, 428 Histological Stains, 428 Protocol PI 8.3 Dilution Cloning in Dishes, 468 Protocol P20.1 20.2.2 20.3 Soft Agar, 473 Protocol P20.2 20.3.2 References, 430 Adherent Clones, 477 Protocol P20.4 Cell Counting hy Hernocytometer, 440 19.1.2 19.1.3 19.1.4 19.2 Automated Cell Counting, 442 Counting Adherent Cells, 446 Cell Weight and Packed Cell Volume (PCV), 446 20.5 20.6 19.2.2 Dye Uptake Assays, 449 20.6.2 20.6.3 20.7 20.7.4 19.4 Cloning Efficiency, 456 19.4.1 Conclusions: Cloning and Selection, 487 Suppliers, 487 References, 487 Clonogenic Assay for Attached Cells, 457 19.5 Selective Media and Inhibitors, 485 Selective Substrates, 486 Selection by Adhesion and Detachment, 486 Selection by Anchorage-Independent Growth, 486 Clonogenic Assays, 456 Protocol P'19.4 19.4.2 19.4.3 20.8 Preparation of
Feeder Layers, 483 Optimization of Clonal Growth, 484 Selective Culture Conditions, 485 20.7.1 20.7.2 20.7.3 Generating a Growth Curve Using Parameters Derived from the Growth Curve, 455 Cloning on Feeder Layers, 482 Protocol P20.7 Multiwell Plates, 452 19.3.3 Preparation of Conditioned Medium, 481 The Growth Curve, 451 Experimental Design, 452 Protocol PI9.3 Cloning Using Conditioned Medium, 481 Protocol P20.6 Cell Proliferation, 450 19.3.1 19.3.2 Replica Plating, 480 Stimulation of Cloning Efficiency, 481 20.6.1 Cell Counting Using Trypan Blue, 448 Isolation of Suspension Clones, 480 Dye Exclusion Assays, 448 Protocol P19.2 Suspension Clones, 479 Protocol P20.5 Cell Viability, 448 19.2.1 19.3 20.4.2 Manual Cell Counting, 438 Protocol PI 9.1 Isolation of Adherent Clones with Cloning Rings, 477 Cell Counting, 437 19.1.1 Cloning in Methocel, 476 Selection of Clones, 477 20.4.1 19. QuantitatiQn and Growth Kinetics, 43] Cloning in Agar, 474 Methylcellulose (Methocel), 476 Protocol P20.3 20.4 Dilution Cloning, 468 Dilution Cloning in Microwell Plates, 472 Cloning in Suspension, 473 20.3.1 18.5.3 Immunocytochemistry, 430 Suppliers, 430 19.1 Colony Counting, 459 Analysis of Colony Formation, 459 DNA Synthesis, 460 XIII 21. Cell Separation and Sorting, 491 21.1 Cell Density and Isopycnic Centrifugation, 491
XIV Contents 21.1.1 22.4.2 22.4.3 Toxicity, 527 Indels and Rearrangements at the Target Site, 527 22.4.4 Off-Target Effects, 527 22.4.5 Oncogenesis, 528 Suppliers, 528 Density Gradient Centrifugation, 491 Protocol P2Í.Í Cell Separation by Centrifugation on a Density Gradient, 493 21.1.2 21.2 Cell Size and Sedimentation Velocity, 495 21.2.1 21.2.2 21.3 Optimization of Density Gradients, 495 Velocity Sedimentation at Unit Gravity, 495 Centrifugal Elutriation, 496 Magnetic Separation and Sorting, 496 Protocol P2Í.2 Magnet-Activated Cell Sorting (MACS), 499 21.4 21.5 Fluorescence-Activated Cell Sorting (FACS), 500 Microfluidic Sorting, 502 Minireview М2 І A 21.6 Microfluidic Cell Culture, 503 Conclusions: Sorting and Separation, 505 Suppliers, 505 PARI VII Stem Cells and Differentiated Cells, 035 23. Culture of Stem Cells, 537 23.1 23.2 References, 505 . 23.3 Gene Delivery, 509 22.1.1 23.4 Transfection with Calcium Phosphate, 511 22.1.3 22.1.4 22.2 22.2.1 22.2.2 22.2.3 Zinc Finger Nucleases (ZFNs), 517 Transcription Activator-Like Effector Nucleases (TALENs), 517 CRISPR/Cas RNA-Guided Nucleases, 519 Cryopreservation and Thawing, 554 23.5 23.6 23.7 23.8 Perinatal Stem Cells, 556 Adult Stem Cells, 557 Stem Cell Characterization and Banking, 558 Conclusions: Culture of Stem Cells, 560 Suppliers, 561 References, 561 Immortalization, 523 22.3.1 22.3.2 22.3.3 Early Immortalization Strategies, 523 Immortalization Using Viral Genes and Oncogenes, 523 Immortalization Using Telomerase, 524 Protocol P22.4 Immortalization Using ItTERT
Transfection, 524 22.3.4 22.4 Evolution in Culture ofhPSCs, 549 Culture Conditions, 550 Feeding and Subculture, 551 Protocol P23.3 Cryopreservation Using ROCK Inhibitor, 555 Electroporation, 514 Viral Transduction, 514 Protocol P22.3 Delivery of CRISPR/Cas9 RNP Using Electroporation, 522 22.3 23.4.4 Optimization of Lipofection, 513 Gene Editing, 517 Human Pluripotent Stem Cell (hPSC) Lines, 549 Protocol P23.2 Subculture in Chemically Defined Conditions, 551 Transfection with Cationic Lipids and Polymers, 512 Protocol P22.2 Induction of Pluripotency, 545 23.4.1 23.4.2 23.4.3 Protocol P22.1 Calcium Phosphate Coprecipitation, 511 22.1.2 Mouse (mESCs), 540 Human (hESCs), 542 Other Species, 545 Protocol P23.1 Generation of iPSCs Using Sendai Viral Vectors, 547 Immortalization, 509 22.1 Terminology: Stem Cells, 537 Embryonic Stem Cells (ESCs), 540 23.2.1 23.2.2 23.2.3 22 5^4012011^7084 .“ References, 528 Conditional Reprogramming, 525 Screening and Artifacts, 526 22.4.1 Selection of Modified Cells, 526 24. Culture uf Specific Cell Types, 507 24.1 24.2 Specialized Cells and Their Availability, 567 Epithelial Cells, 572 24.2.1 24.2.2 24.2.3 24.2.4 Epidermis, 572 Cornea, 573 Oral Epithelium, 573 Bronchial and Tracheal Epithelium, 574
Contents 24.2.5 Gastrointestinal Tract, 574 24.2.6 Liver, 574 24.2.7 Pancreas, 574 24.2.8 Breast, 575 24.2.9 Cervix, 577 24.2.10 Prostate, 577 Mesenchymal Cells, 577 24.3 24.3.1 Connective Tissue, 577 24.3.2 Adipose Tissue, 578 24.3.3 Muscle, 579 24.3.4 Cartilage, 579 24.3.5 Bone, 579 24.3.6 Endothelium, 580 24.4 Neuroectodermal Cells, 580 24.4.1 Neurons, 580 24.4.2 Glial Cells, 580 24.4.3 Endocrine Cells, 581 24.4.4 Melanocytes, 581 24.5 Hematopoietic Cells, 581 24.5.1 Lymphoid Cells, 581 24.5.2 Macrophages and Myeloid Cells, 582 24.5.3 Erythroid Cells, 583 24.5.4 Hybridoma Cells, 583 24.5.5 Chimeric Antigen Receptor (CAR) Т-Cells (CAR T-Cells), 584 24.6 Culture of Cells from Poikilotherms, 585 24.6.1 Fish Cells, 586 24.6.2 Insect Cells, 586 Suppliers, 587 References, 587 25. Culture of Tumor Cells, 593 25.1 25.2 25.3 25.4 Challenges of Tumor Cell Culture, 593 Primary Culture of Tumor Cells, 594 25.2.1 Selection of Representative Cells, 594 Protocol P25.1 Freezing Tumor Biopsies, 594 25.2.2 Disaggregation of Tumor Samples, 596 Development of Tumor Cell Lines, 596 25.3.1 Subculture of Primary Tumor Cultures, 596 25.3.2 Continuous Tumor Cell Lines, 596 25.3.3 Validation ofTumor Cell Lines, 598 25.3.4 Characterization ofTumor Cell Lines, 598 Selective Culture of Tumor Cells, 599 25.4.1 Selective Media and Techniques, 599 25.4.2 Confluent Feeder Layers, 599 Protocol P25.2 Growth on Confluent Feeder Layers, 600 25.4.3 Suspension Cloning, 601 25.4.4 Spheroid Culture, 603 25.4.5 Xenografts, 603 XV 25.5 Specific Tumor Types, 603 25.5.1 Carcinoma, 604 25.5.2 Sarcoma, 606 25.5.3
Melanoma, 606 25.5.4 Lymphoma and Leukemia, 606 25.5.5 Glioma, 606 25.6 Cancer Stem Cells (CSCs), 606 Minireview M25.1 Culture of Cancer Stem Cells, 606 Suppliers, 608 References, 608 26. Differentiation, 616 In Vitro Models of Differentiation, 615 Differentiation Status in Culture, 617 26.2.1 Differentiation and Malignancy, 617 26.2.2 Proliferation and Differentiation, 618 26.2.3 Dedifferentiation, 618 26.2.4 Transdifferentiation, 618 26.2.5 Epithelial-Mesenchymal Transition (EMT), 619 26.3 Induction of Differentiation, 620 26.3.1 Exogenous Soluble Factors, 620 26.3.2 Endogenous Factors, 622 26.3.3 Geometry and Polarity, 625 26.3.4 Cell-Cell Interactions, 625 26.3.5 Cell-Extracellular Matrix (ECM) Interactions, 626 26.3.6 Air-Liquid Interface, 628 26.3.7 Biomechanical Regulation, 628 26.4 Practical Aspects, 628 26.5 Ongoing Challenges, 629 26.5.1 Markers of Differentiation, 629 26.5.2 Stem Cell Differentiation, 629 Suppliers, 631 References, 631 26.1 26.2 PARI VIII Model Environmentsand Applications, 639 11 Three-Dimensional Coltore, 641 . 27.1 27.2 27.3 Terminology: 3D Culture, 641 Technologies for 3D Culture, 643 Minireview М27.1 Advances in Technologies Enabling 3D Cell Culture and the Formation of Tissue-Like Architecture In Vitro, 643 Benefits and Limitations of 3D Culture, 646
XVI Contents 27.4 Scaffold-Free 3D Culture Systems, 647 27.4.1 Spheroid Culture, 647 Protocol P27.1 Tumor Spheroid Formation and Embedding, 649 27.4.2 Dynamic Culture Systems, 650 27.5 Scaffold-Based 3D Culture Systems, 652 27.5.1 Overlay, Embedding, and Encapsulation, 652 27.5.2 Filter Well Inserts, 655 Protocol P27.2 Culture Using Filter Well Inserts, 65 7 27.5.3 Hollow Fiber Systems, 658 27.5.4 Microcarriers and Macrocarriers, 658 27.6 Organoid Culture, 659 27.7 Organotypic Culture, 660 27.7.1 Tissue Equivalents, 660 27.7.2 Tissue Engineering, 660 27.8 Organ Culture, 662 27.9 Characterization of 3D Cultures, 662 Suppliers, 663 References, 663 28. Scale-Up anti Automation, 669 28.1 Terminology: Scale-Up and Bioreactors, 669 28.2 Scale-Up in Suspension, 671 28.2.1 Spinner Culture, 671 28.2.2 Single-Use Bioreactor Systems, 673 28.2.3 Scaffold-Free Perfusion Bioreactors, 675 28.2.4 Other Bioreactor Systems for Suspension Culture, 675 28.3 Scale-Up in Monolayer, 677 28.3.1 Roller Culture, 677 28.3.2 Multisurface Propagators, 678 Protocol P28.1 Handling a Nunc Cell Factory, 678 28.3.3 Microcarrier Culture, 681 28.3.4 Scaffold-Based Perfusion Bioreactors, 684 28.4 Monitoring and Process Control, 685 28.5 Scale-Up for Manufacture, 688 Minireview M28. Í Culture Scale-Up and Bioreactors, 688 28.6 High-Throughput Screening, 691 28.7 Automation and Bioprinting, 691 28.7.1 Automation of Culture Handling, 692 28.7.2 Automation of Cell-Based Assays, 692 28.7.3 3D Bioprinting, 694 Suppliers, 696 References, 696 29. Toxicity Testing, 701 29.1 In Vitro Toxicity Testing, 701 29.1.1
Applications, 702 29.1.2 Limitations, 703 29.1.3 Requirements, 703 29.2 Cytotoxicity Assays, 704 29.2.1 Selecting a Cytotoxicity Assay, 704 29.2.2 Assays Based on Cell Metabolism, 704 Protocol P29. Í MTT-Based Cytotoxicity Assay, 707 29.2.3 Assays Based on Cell Death, 709 29.2.4 Assays Based on Cell Survival, 710 29.2.5 Analysis of Cytotoxicity Assays, 711 29.3 Genotoxicity Assays, 715 29.4 Carcinogenicity Assays, 716 29.5 Advanced Models for Toxicity Testing, 716 29.5.1 3D Models for Eye and Skin Irritation, 716 29.5.2 Organ-on-Chip Technologies, 717 Suppliers, 719 References, 719 PART IX Teaching and Troubleshooting, 728 30. Training, 727 30.1 Training Principles, 727 30.1.1 Roles and Responsibilities, 727 30.1.2 Induction, 728 30.1.3 Training Documents, 729 30.1.4 Hands-on Training, 729 30.2 Training Programs, 729 30.2.1 Topics, 729 30.2.2 Exercises, 731 References, 731 31. Problem Solving, 733 31.1 31.2 31.3 31.4 Microbial Contamination, 733 31.1.1 Type of Microbial Contamination, 734 31.1.2 Contamination Is Limited to One Person, 734 31.1.3 Contamination Is Widespread, 736 31.1.4 Problems with Laminar Flow or Air Quality, 737 Cross-Contamination and Misidentification, 737 Chemical Contamination, 738 Slow Cell Growth, 738
Contents 31.4.1 Problem Is Limited to One Person, 739 31.4.2 Problem Is Widespread, 739 31.5 Abnormal Cell Appearance, 740 31.6 Problems with Materials, 741 31.6.1 Culture Vessels, 741 31.6.2 Medium Formulation and Preparation, 741 31.6.3 Medium Stability and Storage, 743 31.6.4 Water Quality, 743 31.7 Problems with Primary Culture, 744 31.7.1 Suspected Contamination, 744 31.7.2 Poor Take in Primary Culture, 744 31.7.3 Incorrect Phenotype after Primary Culture, 745 31.8 Problems with Feeding or Subculture, 746 31.8.1 pH after Feeding, 746 31.8.2 Poor Take after Subculture, 746 31.8.3 Uneven Growth after Subculture, 747 31.9 Problems with Cryopreservation, 748 31.9.1 Loss of Frozen Stocks, 748 31.9.2 Poor Viability after Thawing, 749 31.9.3 Changed Appearance after Thawing, 750 31.10 Problems with Cloning, 750 31.10.1 Too Few Colonies per Dish, 750 31.10.2 Too Many Colonies per Dish, 751 31.10.3 Non-Random Distribution of Colonies, 751 31.10.4 Incubation of Cloning Dishes, 752 References, 752 32. In Conclusion, 753 Appendix A Appendix В Glossary, 755 Calculations and Preparation of Reagents, 761 Calculations, 761 Counting Cells with a Hemacytometer, 761 Dilution of a Cell Suspension, 761 Population Doubling Level (PDL), 761 Molarity, 762 Percentages and Dilutions, 762 Presane, 762 kotor Speed (rpm to g), 762 XVII Preparation of Reagents, 762 Acetic Acid: Methanol, 762 Agar (2.5%), 762 Alcohol (70%), 762 Bacto™ Peptone (5%), 763 Balanced Salt Solutions, 763 Carboxymethylcellulose (CMC; 4%), 763 Chick Embryo Extract, 763 Collagénase, 763 Collection Medium, 763 Crystal Violet
(0.1%), 764 Dexamethasone (1 mg/ml), 764 Dissection Balanced Salt Solution (DBSS), 764 Dulbecco’s Phosphate-Buffered Saline Without Ca2+ and Mg2+ (DPBS-A), 764 ED7A (10 mM in DPBS-A), 764 EGTA, 764 Erythrosin В, 764 Gelatin (1%), 765 Giemsa Stain, 765 Glucose (20%), 765 Glutamine, 200 mM, 765 Hanks’s Balanced Salt Solution (HBSS), 765 HAT Medium, 765 HB Medium, 765 HEPES, 765 Hoechst 33258, 766 Media, 766 2-Mcrcaptocthanol (ß-Mercaptoethanol; 0.1 M), 766 Methylcellulose (Methocel, 1.6%), 766 Mitomycin C (100 pg/ml), 766 MTT (50 mg/ml), 766 N2 Supplement, 766 N2B27 Medium, 767 Naphthalene Black (Amido Black; 1%), 767 Non-essential Amino Acids (NEAA, lOOx), 767 Paraformaldehyde (4%), 767 Trypan Blue (0.4%), 767 Trypsin (2.5%), 768 Versene, 768 Suppliers, 768 References, 768 Appendix C Media Formulations, 769 References, 779 Index, 781 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Capes-Davis, Amanda |
| author_GND | (DE-588)1112631836 (DE-588)1055981578 |
| author_facet | Capes-Davis, Amanda |
| author_role | aut |
| author_sort | Capes-Davis, Amanda |
| author_variant | a c d acd |
| building | Verbundindex |
| bvnumber | BV047205308 |
| classification_rvk | WC 5100 WX 6600 WX 6603 |
| ctrlnum | (OCoLC)1252697684 (DE-599)BVBBV047205308 |
| discipline | Biologie |
| discipline_str_mv | Biologie |
| edition | Eighth edition |
| format | Book |
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| id | DE-604.BV047205308 |
| illustrated | Illustrated |
| index_date | 2024-07-03T16:52:27Z |
| indexdate | 2024-07-10T09:05:36Z |
| institution | BVB |
| isbn | 9781119513018 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-032610228 |
| oclc_num | 1252697684 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-703 DE-634 DE-11 DE-188 |
| owner_facet | DE-355 DE-BY-UBR DE-703 DE-634 DE-11 DE-188 |
| physical | xxix, 796 Seiten Illustrationen, Diagramme |
| publishDate | 2021 |
| publishDateSearch | 2021 |
| publishDateSort | 2021 |
| publisher | Wiley Blackwell |
| record_format | marc |
| spelling | Capes-Davis, Amanda Verfasser (DE-588)1112631836 aut Freshney's culture of animal cells a manual of basic technique and specialized applications Amanda Capes-Davis, R. Ian Freshney ; reviewing editors: Robert J Geraghty, Raymond W. Nims Culture of animal cells Eighth edition Hoboken, NJ, USA Wiley Blackwell [2021] © 2021 xxix, 796 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier "A companion website with additional resources is available at: www.wiley.com/go/freshney/cellculture8." - Rückseite Cover Methode (DE-588)4038971-6 gnd rswk-swf Zellkultur (DE-588)4067547-6 gnd rswk-swf Tiere (DE-588)4060087-7 gnd rswk-swf Zellkultur (DE-588)4067547-6 s Tiere (DE-588)4060087-7 s DE-604 Methode (DE-588)4038971-6 s Freshney, Robert Ian 1938-2019 Begründer eines Werks (DE-588)1055981578 oth Erscheint auch als Online-Ausgabe, PDF 978-1-119-51303-2 Erscheint auch als Online-Ausgabe, EPUB 978-1-119-51304-9 Digitalisierung UB Regensburg - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032610228&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Capes-Davis, Amanda Freshney's culture of animal cells a manual of basic technique and specialized applications Methode (DE-588)4038971-6 gnd Zellkultur (DE-588)4067547-6 gnd Tiere (DE-588)4060087-7 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4067547-6 (DE-588)4060087-7 |
| title | Freshney's culture of animal cells a manual of basic technique and specialized applications |
| title_alt | Culture of animal cells |
| title_auth | Freshney's culture of animal cells a manual of basic technique and specialized applications |
| title_exact_search | Freshney's culture of animal cells a manual of basic technique and specialized applications |
| title_exact_search_txtP | Freshney's culture of animal cells a manual of basic technique and specialized applications |
| title_full | Freshney's culture of animal cells a manual of basic technique and specialized applications Amanda Capes-Davis, R. Ian Freshney ; reviewing editors: Robert J Geraghty, Raymond W. Nims |
| title_fullStr | Freshney's culture of animal cells a manual of basic technique and specialized applications Amanda Capes-Davis, R. Ian Freshney ; reviewing editors: Robert J Geraghty, Raymond W. Nims |
| title_full_unstemmed | Freshney's culture of animal cells a manual of basic technique and specialized applications Amanda Capes-Davis, R. Ian Freshney ; reviewing editors: Robert J Geraghty, Raymond W. Nims |
| title_short | Freshney's culture of animal cells |
| title_sort | freshney s culture of animal cells a manual of basic technique and specialized applications |
| title_sub | a manual of basic technique and specialized applications |
| topic | Methode (DE-588)4038971-6 gnd Zellkultur (DE-588)4067547-6 gnd Tiere (DE-588)4060087-7 gnd |
| topic_facet | Methode Zellkultur Tiere |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032610228&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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