Basic cell culture : a practical approach / edited by J.M. Davis.

Format:

Book

Contributor:

Davis, J. M. (John M.)

Series:

Practical approach series ; 254.

The practical approach series ; no. 254

Language:

English

Subjects (All):

Cell culture--Laboratory manuals.

Cell culture.

Genre:

Laboratory manuals.

Physical Description:

xxv, 381 pages : illustrations ; 26 cm.

Edition:

Second edition.

Place of Publication:

Oxford ; New York : Oxford University Press, 2002.

Summary:

The use of animal, including human, cell culture has expanded enormously during the last twenty years, with the new applications appearing all the time. This book guides the newcomer progressively through all those areas which are basic to the performance of cell culture. It will also prove useful to the experienced worker entering a new field or setting up a new laboratroy, and as a source of reference on basic techniques. The topics covered include setting up and equipping a cell culture laboratory, sterilization of fluids and equipment, culture media, basic culture technique and the maintenance of cell lines, primary culture and the isolation of new cell lines, specific cell types and their requirements, single cell cloning, quality control of cell lines and the prevention, detection, and cure of contamination.

Contents:

1 The cell culture laboratory / Caroline B. Wigley 1

1 Animal cell culture: past, present, and future 1

2 The laboratory 3

Design concepts and layout 3

Services 8

3 Equipping the laboratory 10

Major items of equipment and instrumentation 10

Culture plasticware and associated small consumable items 18

4 Washing reusable tissue culture equipment 22

Soaking 23

Washing 23

Pipette washing 24

5 General care and maintenance of the tissue culture laboratory 25

2 Sterilization / Peter L. Roberts 29

What is sterilization? 29

The importance of sterility in cell culture 29

The use of antibiotics 30

2 Basic principles 30

3 Wet heat at up to 100[degree]C 31

Theory 31

Pasteurization 31

100[degree]C 32

4 Wet heat above 100[degree]C and autoclaving 32

Theory 32

Steam 33

Types of autoclave 33

Preparation of the load and operation of the autoclave 37

Autoclave testing 40

5 Dry heat 43

Incineration 43

Hot-air ovens 44

6 Irradiation 47

Ultraviolet light 47

Gamma rays 47

7 Chemical sterilization 48

Fumigation 48

Liquid disinfectants 50

8 Filtration 52

Filters for bacteria and fungi 52

Filters for viruses 58

HEPA filters 60

9 Viruses and prions 62

Virus elimination 62

Prions 63

10 Safety in the laboratory 65

11 Good Manufacturing Practice and Good Laboratory Practice 65

3 Culture media / T. Cartwright, G. P. Shah 69

2 Basal media 70

Types of basal media 70

Constituents of basal media 71

Choice of basal medium 74

Preparation of basal medium 75

3 Serum 78

Why use serum? 78

Types of serum 79

Constituents of serum 79

Potential problems with the use of serum 81

Sourcing and selection of serum 85

Serum storage and use 87

4 Replacement of serum in media 88

Reduced-serum media for early-passage normal human cells 88

Serum-free media (SFM) 90

Design of SFM 92

5 Influence of cell culture systems on the choice of medium 98

Batch or perfusion cultures 98

Anchorage-dependent cells 98

Stirred suspension cultures 99

High density culture systems 100

Very low density cultures 102

6 In-house medium development and production versus commercial supply 102

Economic considerations 102

Development of a dedicated medium 103

Quality assurance and control 103

4 Microscopy of living cells / Ian Dobbie, Daniel Zicha 107

2 The modern light microscope 108

Basic scheme 108

Properties of light 109

Magnification 110

Resolution 110

Aberrations and their corrections 113

Illumination 115

Maintenance 118

3 Microscopic techniques 119

Bright field 119

Oblique illumination 119

Dark field 119

Phase-contrast 119

Differential interference contrast (DIC) 120

Hoffman modulation contrast 121

Reflection contrast 121

Varel contrast 122

Polarizing microscopy 122

Interference microscopy 123

Fluorescence microscopy 123

4 Specimen preparation 125

Culture flasks and Petri dishes 125

Coverslips 126

Chambers 127

Interaction with microscopic techniques 128

5 Environmental control for microscopic specimens 128

6 Recording of microscopic images 129

Time-lapse imaging 129

7 Analysis of microscopic images 130

Image enhancement by deconvolution 131

Image processing

extraction of morphometric data 131

Data analysis

statistical evaluation 131

Data interpretation

simulation modelling 132

5 Basic cell culture technique and the maintenance of cell lines / James A. McAteer, John M. Davis 135

The terminology of cell and tissue culture 135

2 Basic components of the cell culture environment 139

Culture medium 140

Physicochemical factors 140

Stationary versus dynamic media supply 140

3 Sterile technique and contamination control 141

Working within the laminar flow hood 142

Pipetting and prevention of aerosol formation 143

Additional considerations for good sterile technique 144

Prevention of cellular cross-contamination 145

Cell culture at the open bench 145

4 General procedures for the cell culture laboratory 146

Maintenance of the laboratory 146

Daily inspection of ongoing cultures 148

Maintenance of stock cultures 148

5 The culture of attached cells 149

Routine culture substrates 150

Choice of culture vessels 152

Artefacts of cell attachment and growth 155

Use of attachment factors and bio-substrates 156

Alternative culture substrates 159

Three-dimensional matrices 159

Culture of cells on microcarriers 160

Mass culture systems for adherent cells 161

6 The culture of cells in suspension 162

Non-adherent substrates for small-scale culture 162

Mass culture of cells in fluid suspension 162

Micro-encapsulation 163

7 In vitro cell growth behaviour 163

Adaptation to culture 163

Phases of cell growth 164

8 Determinations of cell growth data 165

Calculation of in vitro age 165

Multiplication rate and population doubling time 167

Counting cells in suspension 167

Counting cells adherent to a substrate 171

Phases of the cell cycle 173

Cell generation time by time-lapse photomicrography 175

Growth fraction 175

Expressions of culture 'efficiency' 175

9 Cryopreservation and retrieval of cells from frozen storage 176

Purpose of cell banking 176

Mechanism of cell freezing, and factors that affect viability 176

Supplies and equipment for cell freezing 177

Additional comments regarding cryopreservation 179

10 Transportation of cells 181

Transporting frozen cells 181

Transporting growing cells 182

11 Safety in the cell culture laboratory 183

Potential risks in routine cell culture 183

Awareness of increased risk associated with human cells 184

Classification of cell lines as aetiological agents 184

Precautions in handling pathogenic organisms and human cells 185

6 Primary culture and the establishment of cell lines / Caroline MacDonald 191

2 Establishment of primary cultures from various sources 191

Source of material 191

Isolation of cells 193

Substrate for attachment 198

Culture conditions 199

3 Evolution of primary cultures 201

Subculture 201

Growth phases 202

4 Characteristics of limited lifespan cultures 202

Lifespan and senescence 202

Phenotype 203

Karyotype 203

5 Establishment of continuous cell lines 205

Spontaneous 205

Chemical transformation 205

Viral transformation 206

6 Properties of continuous cell lines 206

Aneuploidy 206

Heterogeneity and instability 207

Differentiated status 207

Tumorigenicity 207

7 Cell fusion 207

Methods 207

Properties of hybrids 209

Applications 213

8 Genetic engineering techniques 214

Introduction of genes 214

Oncogene immortalization 218

9 Safety considerations 220

Advantages of cell culture over in vivo experimentation 222

Limitations 222

7 Specific cell types and their requirements / J. D. Sato, D. W. Barnes, I. Hayashi, J. Hayashi, H. Hoshi, T. Kawamoto, W. L. McKeehan, R. Matsuda, K. Matsuzaki, T. Okamoto, G. Serrero, D. J. Sussman, M. Kan 227

3 Growth requirements of cells in vitro 229

Epithelial cells 229

Mesenchymal cells 239

Neuro-ectodermal cells 248

Gonadal cells 250

Embryonal stem (ES) cells 253

Leukocytes 256

Insect cells 262

8 Cloning / John Clarke, Alison J. Porter, Robin Thorpe, John M. Davis 275

Development of techniques 275

Uses of cloning 276

Limitations of cloning 276

2 Special requirements of cells growing at very low densities 278

3 Cell cloning procedures 279

Choice of technique 279

Methods applicable to both attached and suspension cells 280

Methods for attached cells 287

Methods for suspension cells 290

9 The quality control of cell lines and the prevention, detection, and cure of contamination / B. J. Bolton, P. Packer, A.

Doyle 295

2 Obtaining the basic material 296

Importance of cell culture collections 296

Resource centres 297

Cell culture databases 297

3 Quarantine and initial handling of cell lines 297

Accessioning scheme 298

Laboratory design 299

Handling of cell lines 299

Production of cell banks 299

4 Microbial quality control 300

Sources of microbial contamination 300

Testing for bacteria, yeasts, and other fungi 301

Testing for mycoplasma 302

Virus testing 307

Bovine spongiform encephalopathy 313

Elimination of contamination 313

5 Authentication 314

Isoenzyme analysis 315

Cytogenetic analysis 316

DNA fingerprinting 317

6 Regulatory aspects 320

10 Good Laboratory Practice in the cell culture laboratory / Barbara Clitherow, Stephen J. Froud, Jan Luker 325

1 What happens if you fall under a bus? 325

2 GLP, an overview 326

3 What is GLP and when should it be used? 327

4 Planning the study 328

5 Performance of the study 329

Personnel 329

Methods 330

Equipment 331

Materials 332

6 Monitoring 333

Facilities and systems 333

Study-specific audits 333

Final report audit 334

7 Records 334

8 Advice for researchers creating new cell lines 335.

Notes:

First edition published by IRL Press, 1994.

Includes bibliographical references and index.

ISBN:

0199638543

0199638535

OCLC:

47716359