Animal cell

culture
a practical approach
Edited by
RI Freshney
Cancer Research Campaign, Department of Oncology,
University of Glasgow, 1 Horselethill Road,
Glasgow, G12 9LX, UK

FACHBEREICH B I O L O G I E (10)
der Technischen Hochschule Darmstadt
- Brbliothek D - 6 1 0 0 D a r m s t a d t / B. R. D.
Schnlttsoorinstrafie

OIRL PRESS
Oxford Washington DC

Contents
ABBREVIATIONS
1.

xiii

INTRODUCTION: PRINCIPLES OF STERILE TECHNIQUE

AND CELL PROPAGATION
R.I. Freshney
Introduction
Biology of Cells in Culture
Origin and characterisation
Differentiation
Choice of Materials
Organ culture or cell culture?
Source of tissue
Subculture
Selection of medium
Gas phase
Substrate
Preparation
Substrate
Medium
Cells
References

..

.
.

2 . TOWARDS CHEMICALLY-DEFINED, SERUM-FREE MEDIA

FOR MAMMALIAN CELL CULTURE
H.R.Maurer
Introduction
Role of Serum in Cell Culture
Role of serum components
Potential advantages of using low-serum and serum-free media
Distinct disadvantages of using serum in cell culture
Potential disadvantages of serum-free"media
Who is mainly interested in serum-free media?
Replacement of Serum in Medium
General procedures
Considerations prior to medium selection
Practical Suggestions for Medium Selection and Handling
General rules and cautions
Strategies of medium optimisation
Commercially Available Media for Optimised Mammalian
Cell Culture

...

1
1
1
2
3
3
3
4
6
6
7
7
7
8
8
11

13

13
13
13
15
20
21
21
21
21
22
26
26
26
28

vn

3.

Procedures for Coating of Culture Surface with Biomatrix

Poly-D-lysine coating of culture surface
Collagen isolation and coating of culture surface
Fibronectin coating of culture surface
References

29
29
29
30
30

SCALING-UP OF ANIMAL CELL CULTURES

B.Griffiths

33

Introduction
General Methods and Culture Parameters
Cell quantification
Equipment and reagents
Practical considerations
Growth kinetics
Medium and nutrients
pH
Oxygen
Types of culture systems
Monolayer Culture
Introduction
Cell attachment
Scaling-up: step 1, roller bottle
Scaling-up: step 2, roller bottle modifications
Scaling-up: step 3, large capacity stationary cultures
Scaling-up: step 4, fermenter systems
Summary
Suspension Culture
Adaptation to suspension culture
Static suspension culture
Small-scale suspension culture
Scaling-up factors
Stirred fermenters
Continuous flow culture
Airlift fermenter
_
Encapsulated cells
References
Appendix
4.

via

33
34
35
36
37
38
39
41
44
46
46
47
48
49
50
51
59
59
60
61
61
61
64
65
67
68
69
69

PRESERVATION AND CHARACTERISATION

R.J.Hay

71

Introduction
Cell Line Banking

71
72

Cell Freezing and Quantitation of Recovery

Equipment
Preparation and freezing .
Reconstruction and quantitating recovery
Cell Line Characteristics
Species verification
Tests for microbial contamination
Testing for intraspecies cross-contamination
Verifying tissue of origin
Identifying and quantitating immunological products
Cell Source Information Banks
CODATA-IUIS hybridoma data bank
MIRDAB
Acknowledgements
References
5.

6.

SEPARATION OF VIABLE CELLS BY CENTRIFUGAL

ELUTRIATION
D.Conkie

73
73
75
76
78
78
84
92
99
104
109
110
111
111
111

113

Introduction
Cell Separation Methods
The Centrifugal Elutriator
Elutriator rotor
Elutriation chamber
Peristaltic pump and additional apparatus
Complete elutriation system
Technical Protocol
Sterilisation of the apparatus
Flow rate calibration
Sample loading
Elutriation of discrete cell populations
Collection and Interpretation of Data
Heterogeneous cell populations (assessment of cell fraction
purity)
Established cell lines (recovery and viability)
Future Development
Acknowledgements
References

113
113
114
114
115
116
118
118
118
119
120
120
121

FLOW CYTOMETRY
L.Morasca and E.Erba

125

Introduction
Design of Equipment

125
126

121
122
123
124
124

IX

Ortho Diagnostic Systems Inc ORTHO CYTOFLUOROGRAF

IIS
Becton Dickinson fluorescence activated cell sorter, FACS 440
Data Output
Descriptive cytograms
Histograms
Computer analysis of data
Preparation of Samples
Cell suspension from different tissues
Examples of Types of Assay
'
Light scatter of unstained cells
Staining procedures
Application of staining procedures
Fluorescence antibody technique using fluorescein
Discussion
Acknowledgements
References
7.

127
130
134
136
136
137
137
137
140
140
141
143
145
146
147
147

ORGAN CULTURE
I.Lasnitzki

149

Introduction
Survey of Organ Culture Techniques
Clotted plasma substrate
Agar substrate
Raft methods
Grid method
Intermittent exposure to medium and gas phase
Watchglass Technique
Preparation of embryo extract
Preparation of chicken plasma
Explantation
The Maximow Single Slide Technique
Materials
Setting up of cultures
.
Renewal of medium
Agar Gel Techniques
Embryological watchglass technique
Embryo Culture for Teratological Studies
Explantation procedure
Gas mixture
Medium
Protocol
Grid Technique
Materials

149
149
150
150
150
150
151
151
151
152
153
156
156
157
157
157
158
160
160
160
160
161
161
162

Explantation procedures
Medium
Explantation
Incubation and perfusion
Renewal of medium
Long-term Cultivation of Human Adult Tissues
Explantation and cultivation
Medium
Incubation
Change of medium
Preparation of Organ Cultures for Light Microscopy
Materials
Fixation dehydration and embedding
Staining methods
Autoradiography
Labelling
Fixation
Dehydration and embedding
Dipping procedure
Guide to Quantitation of Results
Advantages and Limitations of Organ Culture
References
8.

163
165
165
166
167
168
168
168
169
169
171
171
171
171
176
176
177
177
177
179
179
180

CYTOTOXICITY AND VIABILITY ASSAYS

A.P.Wilson

183

Introduction
Background
Specific Techniques
Culture methods
Drug Concentrations
Duration of Drug Exposure
Recovery Period
End-points
Cytotoxicity, viability and survival
- ...
Cytotoxicity and viability
Survival (reproductive integrity)
Assay Comparisons
Technical Protocols
Drugs and drug solutions
Drug incubation
Assay by survival and proliferative capacity
Cytotoxicity assays
Interpretation of Results
Relationship between cell number and cytotoxicity index
Dose-response curves

183
183
184
185
188
189
191
192
192
192
195
196
197
197
200
200
205
209
209
210
xi

Pitfalls and Trouble Shooting

Large standard deviations
Inter-assay variation
Stimulation to above control levels
Cytotoxicity versus anti-neoplastic activity
Automation
Future Developments
References

9.

AUTORADIOGRAPHIC LOCALISATION OF SPECIFIC

CELLULAR RNA SEQUENCES HYBRIDISED IN SITU TO
TRITIUM-LABELLED PROBES
D.Conkie
Introduction
Principle
Advantages of hybridisation in situ
Principal Requirements
.Sequence redundancy
Probe purity
Probe specific activity
Sensitivity
Signal enhancement
Autoradiographic efficiency
Preparation of Reagents and Materials
Materials
General reagents
Hybridisation reagents
Autoradiography
Detailed Protocol: Hybridisation in Situ
Hybridisation in situ
Autoradiography
Kodak AR 10 stripping film autoradiography
Staining stripping film autoradiographs
Liquid emulsion autoradiography
Staining liquid emulsion autoradiographs
~~~~
The Detection of Molecular Hybrids in Cytological Preparations
by Non-radioactive Methods
Acknowledgement
References

212
212
212
212
213
213
214
215

217

217
217
217
217
218
218
218
219
219
220
220
220
220
220
221
221
221
223
223
224
224
224
224
225
225

APPENDICES
I
II

General Reagents
Suppliers of Specialist Items

INDEX
XII

227
229
233