9780470528129

This is the most complete resource on the techniques, equipment, principles, and practices of animal cell culture.

Since publication of the previous edition of this benchmark text, numerous types of equipment and materials have changed and new specialized techniques have been developed, particularly in the area of stem cell research and tissue engineering. These and other developments have been incorporated into this rigorously revised and expanded sixth edition of Culture of Animal Cells, the leading text in the basic methodology of cell culture.

This authoritative volume offers a complete background related to growth of animal cells in culture. It contains many specialized protocols and advice on how to apply some of the basic protocols to teaching cell culture. Beginning with laboratory design, safety, validation, and bioethics, then continuing with preparation of media, primary culture, and cell lines through to characterization and authentication, contamination, specialized techniques, and troubleshooting, the unparalleled coverage includes:

-An all-new section addressing the additional caution required once a primary culture is converted to a cell line

-Differentiation in stem cells and reprogramming of adult cells to form stem cells

-A new section and protocol on cryopreservation by vitrification

-Separate sections dealing with epithelial cells, mesenchymal cells, neurectodermal cells, and hematopoietic cells, a new chapter on germs cells/stemcells/amniocytes

-A new section on organotypic culture

-Increased emphasis on contamination and misidentification including cross contamination with revised protocols for detection and elimination

-A problem-solving chapter, glossary of terms, advice on sourcing materials, and list of suppliers

This landmark text presents the most accessible and comprehensive introduction available to the culture and experimental manipulation of animal cells and is indispensable for those in the field, including clinical and biopharmaceutical researchers and scientists, graduate students, laboratory managers in cell and molecular biology labs and genetics labs, trainees, and technicians.

R. IAN FRESHNEY, PhD, is an honorary Senior Research Fellow in the Centre for Oncology and Applied Pharmacology, part of the Cancer Research UK Beatson Laboratories at the University of Glasgow. He is the author or editor of numerous books and a world-renowned expert on cell culture technique.

List of Figures.

List of Color Plates.

List of Protocols.

Preface and Acknowledgements.

Abbreviations.

1 Introduction.

1.1 Historical Background.

1.2 Advantages of Tissue Culture.

1.3 Limitations.

1.4 Major Differences In vitro.

1.5 Types of Tissue Culture.

2 Biology of Cultured Cells.

2.1 The Culture Environment.

2.2 Cell Adhesion.

2.3 Cell Proliferation.

2.4 Differentiation.

2.5 Cell Signaling.

2.6 Energy Metabolism.

2.7 Origin of Cultured Cells.

3 Laboratory Design, Layout, and Equipment.

3.1 Layout, Furnishing, and Services.

3.2 Layout.

4 Equipment and Materials.

4.1 Requirements of a Tissue Culture Laboratory.

4.2 Aseptic Area.

4.3 Incubation and Culture.

4.4 Preparation and Sterilization.

4.5 Storage.

4.6 Supplementary Laboratory Equipment.

4.7 Specialized Equipment.

5 Aseptic Technique.

5.1 Objectives of Aseptic Technique.

5.2 Elements of Aseptic Environment.

5.3 Sterile Handling.

5.4 Standard Procedure.

5.5 Apparatus and Equipment.

6 Safety, Bioethics, and Validation.

6.1 Laboratory Safety.

6.2 Risk Assessment.

6.3 Standard Operating Procedures.

6.4 Safety Regulations.

6.5 General Safety.

6.6 Fire.

6.7 Ionizing Radiation.

6.8 Biohazards.

6.9 Bioethics.

6.10 Quality Assurance.

6.11 Validation.

7 Culture Vessels and Substrates.

7.1 The Substrate.

7.2 Treated Surfaces.

7.3 Choice of Culture Vessel.

7.4 Specialized Systems.

8 Defined Media and Supplements.

8.1 Development of Media.

8.2 Physicochemical Properties.

8.3 Balanced Salt Solutions.

8.4 Complete Media.

8.5 Serum.

8.6 Selection of Medium and Serum.

8.7 Other Supplements.

9 Serum-Free Media.

9.1 Disadvantages of Serum.

9.2 Advantages of Serum-Free Media.

9.3 Disadvantages of Serum-Free Media.

9.4 Replacement of Serum.

9.5 Selection of Serum-Free Medium.

9.6 Development of Serum-Free Medium.

9.7 Preparation of Serum-Free Medium.

9.8 Animal Protein-Free Media.

9.9 Conclusions.

10 Preparation and Sterilization.

10.1 Preparation of Reagents and Materials.

10.2 Sterilization of Apparatus and Liquids.

10.3 Apparatus.

10.4 Reagents and Media.

10.5 Sterilization of Media.

10.6 Control, Testing, and Storage of Media.

11 Primary Culture.

11.1 Initiation of a Primary Cell Culture.

11.2 Isolation of the Tissue.

11.3 Types of Primary Culture.

12 Subculture and Cell Lines.

12.1 Subculture and Propagation.

12.2 Choosing a Cell Line.

12.3 Routine Maintenance.

12.4 Subculture.

13 Cloning and Selection.

13.1 Cell Cloning.

13.2 Stimulation of Plating Efficiency.

13.3 Suspension Cloning.

13.4 Isolation of Clones.

13.5 Replica Plating.

13.6 Selective Inhibitors.

13.7 Isolation of Genetic Variants.

13.8 Interaction with Substrate.

14 Cell Separation.

14.1 Cell Density and Isopyknic Sedimentation.

14.2 Cell Size and Sedimentation Velocity.

14.3 Antibody-Based Techniques.

14.4 Fluorescence-Activated Cell Sorting.

14.5 Other Techniques.

14.6 Beginner's Approach to Cell Separation.

15 Characterization.

15.1 The Need for Characterization.

15.2 Authentication.

15.3 Record Keeping and Provenance.

15.4 Parameters of Characterization.

15.5 Cell Morphology.

15.6 Confocal Microscopy.

15.7 Chromosome Content.

15.8 DNA Analysis.

15.9 RNA and Protein Expression.

15.10 Enzyme Activity.

15.11 Antigenic Markers.

15.12 Differentiation.

16 Differentiation.

16.1 Expression of the In vivo Phenotype.

16.2 Stages of Differentiation.

16.3 Proliferation and Differentiation.

16.4 Commitment and Lineage.

16.5 Stem Cell Plasticity.

16.6 Markers of Differentiation.

16.7 Induction of Differentiation.

16.8 Differentiation and Malignancy.

16.9 Practical Aspects.

17 Transformation and Immortalization.

17.1 Role in Cell Line Characterization.

17.2 What is Transformation?

17.3 Genetic Instability and Heterogeneity.

17.4 Immortalization.

17.5 Aberrant Growth Control.

17.6 Tumorigenicity.

18 Contamination.

18.1 Sources of Contamination.

18.2 Types of Microbial Contamination.

18.3 Monitoring for Contamination.

18.4 Disposal of Contaminated Cultures.

18.5 Eradication of Contamination.

18.6 Cross-contamination.

18.7 Conclusions.

19 Cryopreservation.

19.1 Rationale for Freezing.

19.2 Considerations before Cryopreservation.

19.3 Principles of Cryopreservation.

19.4 Vitrification.

19.5 Design and Control of Freezer Stocks.

19.6 Cell Banks.

19.7 Transporting Cells.

20 Quantitation.

20.1 Cell Counting.

20.2 Cell Weight.

20.3 DNA Content.

20.4 Protein.

20.5 Rates of Synthesis.

20.6 Preparation of Samples for Enzyme Assay and Immunoassay.

20.7 Cytometry.

20.8 Replicate Sampling.

20.9 Cell Proliferation.

20.10 Plating Efficiency.

20.11 Labeling Index.

20.12 Cell Cycle Time.

20.13 Cell Migration.

21 Cytotoxicity.

21.1 Viability, Toxicity, and Survival.

21.2 In vitro Limitations.

21.3 Nature of the Assay.

21.4 Applications of Cytotoxicity Assays.

21.5 Genotoxicity.

21.6 Inflammation.

22 Specialized Cells.

22.1 Cell Culture of Specialized Cells.

22.2 Epithelial Cells.

22.3 Mesenchymal Cells.

22.4 Neuroectodermal Cells.

22.5 Hematopoietic Cells.

22.6 Gonads.

23 Stem Cells, Germ Cells, and Amniocytes.

23.1 Stem Cells.

23.2 Germ Cells.

23.3 Extraembryonic Cells.

24 Culture of Tumor Cells.

24.1 Problems of Tumor Cell Culture.

24.2 Sampling.

24.3 Disaggregation.

24.4 Primary Culture.

24.5 Selective Culture of Tumor Cells.

24.6 Development of Cell Lines.

24.7 Characterization of Tumor Cell Cultures.

24.8 Specific Tumor Types.

25 Three-Dimensional Culture.

25.1 Cell Interaction and Phenotypic Expression.

25.2 Organ Culture.

25.3 Histotypic Culture.

25.4 Organotypic Culture.

25.5 Imaging Cells in 3-D Constructs.

26 Scale-up and Automation.

26.1 Scale-up in Suspension.

26.2 Scale-up in Monolayer.

26.3 Process Control.

26.4 Automation.

27 Specialized Techniques.

27.1 Lymphocyte Preparation.

27.2 Autoradiography.

27.3 Time-Lapse Recording.

27.4 Cell Synchrony.

27.5 Culture of Cells from Poikilotherms.

27.6 Somatic Cell Fusion.

27.7 Production of Monoclonal Antibodies.

28 Training Programs.

28.1 Objectives.

28.2 Preparative and Manipulative Skills.

28.3 Basic Cell Culture Techniques.

28.4 Advanced Exercises.

28.5 Specialized Exercises.

29 Problem Solving.

29.1 Abnormal Appearance of Cells.

29.2 Slow Cell Growth.

29.3 Medium.

29.4 Substrates and Containers.

29.5 Microbial Contamination.

29.6 Chemical Contamination.

29.7 Primary Culture.

29.8 Differentiation.

29.9 Feeding.

29.10 Subculture.

29.11 Cloning.

29.12 Cross-contamination and Misidentification.

29.13 Cryopreservation.

29.14 Cell Counting.

30 In Conclusion.

Appendix I: Calculations and Preparation of Reagents.

Appendix II: Sources of Equipment and Materials.

Appendix III: Suppliers and Other Resources.

Appendix IV: Glossary.

Appendix V: Cross-contaminated or Misidentified Cell Lines.

Appendix VI: General Textbooks and Relevant Journals.

References.

Index.

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Culture of Animal Cells : A Manual of Basic Technique and Specialized Applications

Culture of Animal Cells : A Manual of Basic Technique and Specialized Applications

0470528125

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Table of Contents

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