Animal Cell Culture A Practical Approach

by Masters, John R. W.

• ISBN13: 9780199637966
• ISBN10: 0199637962
• Edition: 3rd
• Format: Paperback
• Copyright: 2000-08-24
• Publisher: Oxford University Press

Summary

This new edition of Animal Cell Culture covers new or updated chapters on cell authentication, serum-free culture, apoptosis assays, FISH, genetic modification, scale-up, stem cell assays, 3-dimensional culture, tissue engineering and cytotoxicity assays. Detailed protocols for a wide variety of methods provide the core of each chapter, making new methodology easily accessible. Everyone working in biological and medical research, whether in academia or a commercial organization, practicing cell culture will benefit greatly from this book.

Table of Contents

List of protocols	xiii
Abbreviations	xvii
Introduction to basic principles
R. Ian Freshney
Background	1	(1)
Biology of cells in culture	2	(1)
Origin and characterization	2	(1)
Defferentiation	3	(1)
Choice of materials	3	(7)
Cell type	3	(1)
Source of tissue	4	(1)
Subculture	5	(2)
Selection of medium	7	(1)
Gas phase	8	(1)
Culture system	9	(1)
Procedures	10	(9)
Substrate	10	(1)
Medium	11	(1)
Cell culture	11	(5)
References	16	(3)
Scaling-up of animal cell cultures
Bryan Griffiths
Introduction	19	(1)
General methods and culture parameters	20	(12)
Cell quantification	20	(1)
Equipment and reagents	21	(1)
Practical considerations	22	(1)
Growth kinetics	23	(1)
Medium and nutrients	23	(1)
pH	24	(2)
Oxygen	26	(4)
Types of culture process	30	(2)
Summary of factors limiting scale-up	32	(1)
Monolayer culture	32	(16)
Introduction	32	(2)
Cell attachment	34	(1)
Scaling-up	35	(13)
Suspension culture	48	(10)
Adaptation to suspension culture	49	(1)
Static suspension culture	50	(1)
Small scale suspension culture	50	(3)
Scaling-up factors	53	(1)
Stirred bioreactors	54	(2)
Continuous-flow culture	56	(1)
Airlift fermenter	57	(1)
Immobilized cultures	58	(11)
Immurement cultures	59	(3)
Entrapment cultures	62	(1)
Porous carriers	62	(4)
References	66	(3)
Cell line preservation and authentication
R. J. Hay
M. M. Cleland
S. Durkin
Y. A. Reid
Introduction	69	(1)
Cell line banking	70	(1)
Cell freezing and quantitation of recovery	71	(7)
Equipment	72	(1)
Preparation and freezing	73	(1)
Reconstitution and quantitating recovery	74	(4)
Cell line authentication	78	(27)
Species verification	78	(3)
Tests for microbial contamination	81	(13)
Testing for intraspecies cross-contamination	94	(7)
Miscellaneous characterizations and cell line availability	101	(1)
Acknowledgements	102	(1)
References	102	(3)
Development of serum-free media
Soverin Karmiol
Introduction	105	(1)
Role of serum and other undefined tissue extracts in cell culture systems	105	(1)
Response curves	106	(6)
Proliferating cultures	106	(5)
Non-proliferating cultures (hepatocytes)	111	(1)
Antimicrobials, phenol red, Hepes, and light	112	(5)
Phenol red	113	(1)
Gentamicin	114	(1)
Hepes	115	(1)
Light	116	(1)
Purity of components	117	(1)
Fatty acids	117	(6)
Acknowledgements	120	(1)
References	120	(3)
Three-dimensional culture
L. A. Kunz-Schughart
W. Mueller-Kleiser
Introduction	123	(1)
Multicellular tumour spheroids (MCTS)	124	(15)
MCTS monocultures	124	(9)
MCTS co-cultures	133	(6)
Experimental tissue modelling	139	(10)
Current research on tissue modelling	139	(1)
Tissue modelling of skin and mucosa	140	(3)
Embryoid bodies	143	(1)
References	143	(6)
Tissue engineering
Robert A. Brown
Rebecca A. Porter
Introduction	149	(1)
Design stages for tissue engineering	150	(4)
Tissue engineered skin	152	(1)
Tissue engineered urothelium	152	(1)
Tissue engineered peripheral nerve implants	153	(1)
Cell substrates and support materials	154	(3)
Cell sources	157	(2)
Orientation	159	(4)
Mechanical cues	161	(2)
Protocols	163	(12)
Cell seeding of implantable materials	163	(2)
Slow release systems for local control of TE constructs or repair sites	165	(3)
Acknowledgements	168	(1)
References	168	(7)
Cytotoxicity and viability assays
Anne P. Wilson
Introduction	175	(1)
Background	176	(1)
Specific techniques	177	(6)
Culture methods	177	(2)
Duration of drug exposure and drug concentrations	179	(3)
Recovery period	182	(1)
End-points	183	(9)
Cytotoxicity, viability, and survival	183	(1)
Cytotoxicity and viability	183	(8)
Survival (reproductive integrity)	191	(1)
Assay comparisons	192	(1)
Technical protocols	192	(18)
Drugs and drug solutions	192	(2)
Drug incubation	194	(1)
Assay by survival and proliferative capacity	194	(7)
Cytoxicity assays	201	(9)
Interpretation of results	210	(4)
Relationship between cell number and cytoxicity index	210	(1)
Dose-response curves	211	(3)
Pitfalls and troubleshooting	214	(7)
Large standard deviations	214	(1)
Variation between assays	214	(1)
Stimulation to above control levels	214	(1)
References	215	(6)
Fluorescence in situ hybridization
W. Nicol Keith
Introduction	221	(1)
Probes	222	(6)
Probe detection	228	(3)
A final word on the tricky bits	231	(1)
FISH resources	232	(3)
Solutions	232	(1)
Useful books	232	(1)
Useful Web sites	232	(1)
References	233	(2)
Genetic modification
Majid Hafezparast
Introduction	235	(1)
Transfection	235	(10)
Calcium phosphate-DNA co-precipitation	237	(1)
Lipid-mediated gene transfer (lipofection)	238	(2)
Electroporation	240	(3)
Staining of cells for expression of β-galactosidase	243	(1)
Rescue of episomal plasmids	244	(1)
Microcell-mediated chromosome transfer	245	(7)
Formation of micronuclei	246	(1)
Enucleation	247	(1)
Purification of microcells	248	(2)
Fusion of microcells to recipient cells	250	(1)
Selection of microcell hybrids	250	(2)
Irradiation fusion gene transfer	252	(7)
Use in mapping genes	253	(1)
Use in positional cloning	254	(1)
Irradiation doses	255	(1)
References	256	(3)
Epithelial stem cell identification, isolation, and culture
David Hudson
Introduction	259	(1)
Clinical application of cultured human stem cells	259	(1)
Basic principles for identification and purification of stem cells	260	(1)
Assessment of proliferative heterogeneity	260	(5)
Methods for the separation of different cell populations	265	(8)
Isolation of cells by differential adhesion	266	(4)
Separation of cultured and primary cells by flow and immunomagnetic sorting	270	(3)
Long-term maintenance of stem cells in culture	273	(1)
Keratinocyte stem cells in culture	273	(1)
Maintenance of non-epidermal epithelial cells in long-term culture	273	(1)
Stem cell characterization by immunocytochemistry	274	(7)
Antibody markers of differentiated cell phenotypes	274	(3)
Staining cell suspensions using cytospin preparations	277	(1)
References	278	(3)
Senescence, apoptosis, and necrosis
Ian R. Kill
Richard G. A. Faragher
Introduction	281	(1)
Cellular senescence	281	(1)
Cell death	282	(1)
Differentiation and de-differentiation	282	(1)
Simple measures of the population dynamics of primary cultures	282	(9)
Measurement of the growth fraction	283	(4)
Determination of the necrotic fraction of the population	287	(1)
Determination of the senescent fraction of the population	288	(2)
Determination of the apoptotic fraction of the population using TUNEL	290	(1)
Other techniques for analysing population dynamics	291	(12)
Detection of apoptosis using DNA laddering	292	(1)
Inhibition of apoptosis using peptide inhibitors of caspases	293	(2)
Determination of the non-dividing fraction of a population by simplified haptotactic assays, `Ponten Plates'	295	(1)
Determination of telomerase activity and telomere length	296	(5)
Acknowledgements	301	(1)
References	301	(2)
A1 List of suppliers	303	(8)
Index	311