Culture of animal cells: a manual of basic technique
Saved in:
| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
1987
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| Edition: | 2. ed. |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| ISBN: | 0845142410 |
Staff View
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Record in the Search Index
| _version_ | 1804145012317880320 |
|---|---|
| adam_text | Second Edition
CULTURE OF
ANIMAL CELLS
A Manual of
Basic Technique
R Ian_Freshney
Department of Medical Oncology
Cancer Research Campaign Laboratories
University of Glasgow
Alan R Liss, Inc , New York
Contents
PREFACE TO THE SECOND EDITION xv
PREFACE TO THE FIRST EDITION xvii
1 Introduction
BACKGROUND I
ADVANTAGES OF TISSUE CULTURE 3
Control of the Environment 3
Characterization and Homogeneity of Sample 3
Economy 3
DISADVANTAGES 3
Expertise 3
Quantity 4
Instability 4
MAJOR DIFFERENCES IN VITRO 4
Origin of Cells 4
DEFINITIONS 4
2 Biology of the Cultured Cell
THE CULTURE ENVIRONMENT 7
INITIATION OF THE CULTURE 7
EVOLUTION OF CELL LINES 7
CRISIS AND THE DEVELOPMENT OF CONTINUOUS CELL LINES 9
DEDIFFERENTIATE 10
WHAT IS A CULTURED CELL? 12
FUNCTIONAL ENVIRONMENT 12
3 Design and Layout of the Laboratory
STERILE HANDLING AREA 15
Laminar Flow 15
INCUBATION 16
Hot Room 16
SERVICE BENCH 23
PREPARATION 23
WASH-UP 23
STORAGE 23
CONSTRUCTION AND LAYOUT 24
4 Equipping the Laboratory
ESSENTIAL EQUIPMENT 25
Incubator 25
Incubation Temperature 25
Sterilizer 26
Refrigerators and Freezers 26
Microscope 27
Washing-up Equipment 27
Sterilizing and Drying Oven 28
viii Contents
Water Purification 28
Centrifuge 31
Cell Freezing 31
BENEFICIAL EQUIPMENT 32
Laminar Flow Hood 32
Cell Counter 32
Vacuum Pump 32
CO2 Incubator 32
Preparation and Quality Control 33
Upright Microscope 34
Temperature Recording 34
Magnetic Stirrer 34
Roller Racks 34
Pipette Aids and Automatic Pipetting 34
Mechanical Aids and Automation 36
USEFUL ADDITIONAL EQUIPMENT 37
Low-Temperature Freezer 37
Glassware Washing Machine 37
Closed-Circuit TV 37
Colony Counters 38
Cell Sizing 40
Time-Lapse Cinemicrography 40
Controlled-Rate Cooler 40
Centrifugal Elutriator 40
Flow Cytophotometer 40
CONSUMABLE ITEMS 41
Pipettes 41
Culture Vessels 42
5 Aseptic Technique
OBJECTIVES OF ASEPTIC TECHNIQUE 43
QUIET AREA 43
WORK SURFACE 43
PERSONAL HYGIENE 44
PIPETTING T~r ^ 44
STERILE HANDLING 7 7 44
Swabbing 44
Capping 44
Flaming 46
Pouring 46
LAMINAR FLOW 46
STANDARD PROCEDURE 47
6 Laboratory Safety and Biohazards
GENERAL SAFETY 51
Glassware and Sharp Items 51
Chemical Toxicity 51
Gases 52
Liquid N2 52
FIRE 53
RADIATION 53
BIOHAZARDS 55
7 The Culture Environment: Substrate, Gas Phase, Medium, and Temperature
THE SUBSTRATE 57
Glass 57
Disposable Plastic 57
Contents ix
Microcarriers 58
Sterilization of Plastics 38
Alternative Artificial Substrates 58
Treated Surfaces 58
Feeder Layers 60
Three-Dimensional Matrices 60
Nonadhesive Substrates 61
Liquid-Gel or Liquid-Liquid Interfaces 62
Perfused Microcapillary Bundles 62
Culture Vessels 63
THE GAS PHASE 66
Oxygen 66
Carbon Dioxide 67
MEDIA AND SUPPLEMENTS 69
PHYSICAL PROPERTIES 69
pH 69
Preparation of pH Standards 70
Buffering 70
Osmolality 70
Temperature 71
Viscosity 71
Surface Tension and Foaming 71
CONSTITUENTS OF MEDIA 71
Balanced Salt Solutions 71
DEFINED MEDIA 71
Amino Acids 72
Vitamins 72
Salts 72
Glucose 72
Organic Supplements 72
Hormones and Growth Factors 72
SERUM 72
Protein 73
Polypeptides 73
Hormones_ 73
Metabolites and~Nutrients 73
Minerals 73
Inhibitors 73
SERUM-FREE MEDIA 77
Selective Media 79
Disadvantages 79
Replacement of Serum 79
Selection and Development of Serum-Free Medium 80
Preparation of Serum-Free Media 81
Serum Substitutes 81
Conclusions 81
SELECTION OF MEDIUM AND SERUM 81
Batch Reservation 83
Testing Serum 83
OTHER SUPPLEMENTS 83
INCUBATION TEMPERATURE 84
8 Preparation and Sterilization
PROCEDURES FOR THE PREPARATION AND STERILIZATION OF APPARATUS 85
Glassware 85
Pipettes 88
Screw Caps 89
Selection of Detergent 90
x Contents
Miscellaneous Equipment 92
Alternative Methods of Sterilization 93
REAGENTS AND MEDIA 93
Water 93
Balanced Salt Solutions 94
Media 94
Autoclavable Media 96
Filter Sterilization 97
Sterility Testing 99
Culture Testing 100
Storage 101
Serum 102
Preparation and Sterilization of Other Reagents 105
9 Disaggregation of the Tissue and Primary Culture
ISOLATION OF THE TISSUE 107
Mouse Embryos 107
Hen s Egg 110
Human Biopsy Material 112
PRIMARY CULTURE 113
Primary Explant Technique 113
Enzymatic Disaggregation 115
Disaggregation in Warm Trypsin 115
Trypsin at 4°C 117
Chick Embryo Organ Rudiments 118
Other Enzymatic Procedures 122
Collagenase 122
Mechanical Disaggretion 124
Separation of Viable and Nonviable Cells 126
10 Maintenance of the Culture—Cell Lines
NOMENCLATURE 127
ROUTINE MAINTENANCE 128
Replacement~of Medium 130
Volume, Depth, and Surface Area 131
Holding Medium 132
Changing the Medium or Feeding a Culture 132
Subculture 132
Propagation in Suspension 134
SLOW CELL GROWTH 134
11 Cloning and Selection of Specific Cell Types
CLONING 137
Dilution Cloning 137
Stimulation of Plating Efficiency 139
Multiwell Dishes 140
Semisolid Media 140
Isolation of Clones 144
Suspension Clones 147
SELECTIVE MEDIA 147
ISOLATION OF GENETIC VARIANTS 147
INTERACTION WITH SUBSTRATE 151
Selective Adhesion 151
Selective Detachment 151
Nature of Substrate 152
Contents xi
12 Physical Methods of Cell Separation
CELL SIZE AND SEDIMENTATION VELOCITY 155
Unit Gravity Sedimentation 156
Centrifugal Elutriation 160
CELL DENSITY AND ISOPYCNIC SEDIMENTATION 163
FLUORESCENCE-ACTIVATED CELL SORTING 165
OTHER TECHNIQUES 167
13 Characterization
INTRODUCTION 169
Species Identification 169
Lineage or Tissue Markers 169
Unique Markers 170
Transformation 170
MORPHOLOGY 170
Staining 170
Culture Vessels for Cytology - Monolayer Cultures 172
Suspension Culture 172
Photography 175
CHROMOSOME CONTENT 175
Chromosome Preparations 176
Chromosome Banding 176
Trypsin/Giemsa Banding 177
Q-Banding 178
DNA CONTENT 179
RNA AND PROTEIN 179
ENZYME ACTIVITY 180
Isoenzymes 180
ANTIGENIC MARKERS 184
Indirect Immunofluorescence 184
DIFFERENTIATION 185
14 Induction of Differentiation
STAGES OF COMMITMENT AND DIFFERENTIATION 187
PROLIFERATION AND DIFFERENTIATION 187
COMMITMENT AND LINEAGE 189
MARKERS OF DIFFERENTIATION 190
INDUCTION OF DIFFERENTIATION 190
Soluble Inducers 190
Cell Interaction 192
Cell-Matrix Interactions 193
Polarity and Cell Shape 193
DIFFERENTIATION AND MALIGNANCY 193
PRACTICAL ASPECTS 194
Preparation of Collagen Gel 194
15 The Transformed Phenotype
WHAT IS TRANSFORMATION? 197
ANCHORAGE INDEPENDENCE 198
Suspension Cloning 199
Contact Inhibition and Density Limitation of Growth 199
Growth on Confluent Monolayers 201
GENETIC ABNORMALITIES 203
Chromosomal Aberrations 203
CELL PRODUCTS AND SERUM DEPENDENCE 203
xii Contents
Tumor Angiogenesis Factor (TAF) 205
Plasminogen Activator 205
INVASIVENESS 205
TUMORIGENESIS 206
16 Contamination
TYPES OF MICROBIAL CONTAMINATION 207
Monitoring Cultures for Mycoplasmas 207
Fluorescent Technique for Detecting Mycoplasmas 209
Alternative Methods 210
DETECTION OF MICROBIAL CONTAMINATION 211
CROSS CONTAMINATION 212
CONCLUSIONS 214
17 Instability, Variation, and Preservation
ENVIRONMENT 215
SELECTIVE OVERGROWTH, TRANSFORMATION, AND SENESCENCE 215
GENETIC INSTABILITY 215
PRESERVATION 216
Selection of Cell Line 216
Standardization of Medium and Serum 216
Cell Freezing 217
Thawing 221
CELL BANKS 225
18 Quantitation and Experimental Design
CELL COUNTING 227
Hemocytometer 227
Electronic Particle Counting 229
Coulter Counter 229
Operation of Coulter Cell Counter 229
Stained Monolayers 232
CELL WEIGHT 232
DNA CONTENT T-r 233
Determination of DNA Contenttrf Cells by Hoechst 33258 233
Determination of DNA Content of Cells by DAPI 233
PROTEIN 234
Solubilization of Sample 234
Lowry Method 234
Bradford Method 235
Protein Synthesis 235
DNA Synthesis 236
PREPARATION OF SAMPLES FOR ENZYME ASSAY AND IMMUNOASSAY 237
REPLICATE SAMPLING 237
GROWTH CYCLE 238
The Lag Phase 239
The Log Phase 239
The Plateau Phase 240
PLATING EFFICIENCY 241
CLONAL GROWTH ASSAY BY DILUTION CLONING 241
Automatic Colony Counting 241
LABELING INDEX 241
Growth Fraction 243
MITOTIC INDEX 244
CELL CYCLE TIME (GENERATION TIME) 244
CYTOMETRY 244
Contents xiii
19 Measurement of Cytotoxicity and Viability
LIMITATIONS OF IN VITRO METHODS 245
NATURE OF THE ASSAY 245
Short-Term Assays—Viability 246
Long-Term Tests—Survival 247
Metabolic Tests 254
Drug Interaction 254
ANTICANCER DRUG SCREENING 254
Predictive Testing 255
Culture Systems 255
MUTAGENICITY 256
CARCINOGENICITY 256
20 Culture of Specific Cell Types
EPITHELIAL CELLS 257
Epidermis 257
Breast 260
Cervix 261
Gastrointestinal Tract 263
Liver 263
Pancreas 265
Kidney 267
Bronchial and Tracheal Epithelium 267
Prostate 268
MESENCHYMAL CELLS 270
Connective Tissue 270
Adipose Tissue 270
Muscle 270
Cartilage 272
Bone 273
Endothelium 275
NEURECTODERMAL CELLS 277
Neurones 277
Glia _ •• 279
Endocrine Cells 280
Melanocytes 280
HEMOPOIETIC CELLS 284
GONADS 287
MINIMAL DEVIATION TUMORS 287
TERATOMAS 288
21 Culture of Tumor Tissue
SAMPLING 289
DISAGGREGATION 290
PRIMARY CULTURE 290
CHARACTERIZATION 290
DEVELOPMENT OF CELL LINES 291
Continuous Cell Lines 292
GENERAL METHOD 292
SELECTIVE CULTURE 292
Selective Media 292
Selective Substrates 293
Confluent Feeder Layers 293
Suspension Cloning 294
Histotypic Culture 295
xiv Contents
Spheroids 295
Xenografts 295
Freezing 295
22 Three-Dimensional Culture Systems
ORGAN CULTURE 298
Gas and Nutrient Exchange 298
Structural Integrity 298
Growth and Differentiation 299
Limitations of Organ Culture 299
Types of Organ Culture 299
HISTOTYPIC CULTURE 302
Sponge Techniques 303
Capillary Bed Perfusion 303
Reaggregation and Spheroids 304
FILTER WELLS 305
23 Specialized Techniques
MASS CULTURE TECHNIQUES 309
Suspension Culture 309
Monolayer Culture 312
LYMPHOCYTE PREPARATION 318
Blast Transformation 319
AUTORADIOGRAPHY 319
CULTURE OF CELLS FROM POIKILOTHERMS 323
CELL SYNCHRONY 324
Cell Separation 324
Blockade 324
TIME-LAPSE CINEMICROGRAPHY 324
AMNIOCENTESIS 327
SOMATIC CELL FUSION 329
GENE TRANSFER 331
PRODUCTION OF MONOCLONAL ANTIBODIES 334
IN SITU MOLECULAR HYBRIDIZATION 338
VIRUS PREPARATION AND ASSAY 340
IN CONCLUSION 343
REAGENT APPENDIX 345
TRADE INDEX 351
Sources of Materials 351
Addresses of Commercial Suppliers 356
Scientific Societies With Interests in Tissue Culture 361
Late Additions and Alterations to Trade Index 361
GLOSSARY 363
REFERENCES 367
General Textbooks for Further Reading 384
Useful Journals 385
INDEX 387
|
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| spelling | Freshney, Robert Ian 1938-2019 Verfasser (DE-588)1055981578 aut Culture of animal cells a manual of basic technique 2. ed. 1987 txt rdacontent n rdamedia nc rdacarrier Cell Culture cabt Techniques cabt Methode (DE-588)4038971-6 gnd rswk-swf Zellkultur (DE-588)4067547-6 gnd rswk-swf Tierzelle (DE-588)4185509-7 gnd rswk-swf Tiere (DE-588)4060087-7 gnd rswk-swf Gewebekultur (DE-588)4157245-2 gnd rswk-swf Zellkultur (DE-588)4067547-6 s Tiere (DE-588)4060087-7 s Methode (DE-588)4038971-6 s 1\p DE-604 Gewebekultur (DE-588)4157245-2 s 2\p DE-604 Tierzelle (DE-588)4185509-7 s 3\p DE-604 HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=022013973&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 3\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 Cell Culture cabt Techniques cabt Methode (DE-588)4038971-6 gnd Zellkultur (DE-588)4067547-6 gnd Tierzelle (DE-588)4185509-7 gnd Tiere (DE-588)4060087-7 gnd Gewebekultur (DE-588)4157245-2 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4067547-6 (DE-588)4185509-7 (DE-588)4060087-7 (DE-588)4157245-2 |
| title | Culture of animal cells a manual of basic technique |
| title_auth | Culture of animal cells a manual of basic technique |
| title_exact_search | Culture of animal cells a manual of basic technique |
| title_full | Culture of animal cells a manual of basic technique |
| title_fullStr | Culture of animal cells a manual of basic technique |
| title_full_unstemmed | Culture of animal cells a manual of basic technique |
| title_short | Culture of animal cells |
| title_sort | culture of animal cells a manual of basic technique |
| title_sub | a manual of basic technique |
| topic | Cell Culture cabt Techniques cabt Methode (DE-588)4038971-6 gnd Zellkultur (DE-588)4067547-6 gnd Tierzelle (DE-588)4185509-7 gnd Tiere (DE-588)4060087-7 gnd Gewebekultur (DE-588)4157245-2 gnd |
| topic_facet | Cell Culture Techniques Methode Zellkultur Tierzelle Tiere Gewebekultur |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=022013973&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT freshneyrobertian cultureofanimalcellsamanualofbasictechnique |