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1. Animal
Cell Culture
CONCEPT AND APPLICATION
Sheelendra M. Bhatt
Alpha Science International Ltd.
Oxford, U.K.

2. CONTENTS
Preface vii
Abbreviations xvii
1. Developments of the Animal Cell Culture 1.1-1.10
1.1 History 1.1
1.2 Types of Cell Culture 1.3
1.3 Cell Culture and their Scope 1.5
1.4 Properties of the Animal Cells 1.6
1.5 Advantages J
1.6
1.6 Limitations 1.7
1.7 Importance of the Animal Cell Culture 1.8
1.8 Applications ~ 1.8
1.9 In-vitro and In-vivo Culture 1.9
Brain Quest 1.10
2. Laboratory Design 2.1-2.11
2.1 Cell Culture Laboratory 2.1
2.2 Common Culture Instruments 2.3
2.3 Dry Heat Sterilization 2.6
2.4 Moist Heat Sterilization 2.7
2.5 Work Surface and Flooring 2.8
2.6 Plastic Wares and Consumable 2.8
2.7 Care and Maintenance 2.9
2.8 Tissue Culture Grade Water 2.10
Brain Quest 2.11
3. Laboratory Practices , 3.1-3.7
3.1 Containments . 3.1
3.2 Containment Safeguard 3.1
3.3 Laboratory Practices 3.3
3.4 P-4 Level Lab Requirements 3.5
3.5 Handling Frozen Biological Material 3.6
Brain Quest 3.6

3. x Contents
4. Identification and Characterization of Contaminations 4.1-4.10
4.1 Introduction 4.1
4.2 Mycoplasma 4.2
4.3 Viral Infection 4.4
4.4 Fungal Contaminations 4.5
4.5 Antibiotics 4.6
4.6 Characterization and Identification 4.8
4.7 Prevention of Contaminations 4.9
Brain Quest 4.10
5. Sterilization Techniques 5.1-5.7
5.1 Sterilization 5.1
5.2 Sterilization Methods 5.1
5.3 Ionizing Radiation 5.3
5.4 Non-ionizing Radiation 5.3
5.5 Sterilization by Filtration 5.3
5.6 Heavy Metals as Microbicidal Agents 5.5
Brain Quest 5.6
6. Culture Environment 6.1-6.12
6.1 Culture Environment 6.1
6.2 Substrate 6.1
6.3 Mechanism of Cell Adhesion 6.2
6.4 Scale up of Animal Cell Culture 6.3
6.5 Suspension Cultures 6.7
6.6 Fermentation Technology 6.8
6.7 Immobilized Cell Culture 6.11
Brain Quest 6.12
7. Culture Media 7.1-7.13
7.1 History 7.1
7.2 Physiochemical Parameters 7.1
7.3 Media 7.3
7.4 Natural Media 7.4
7.5 Serum Media Composition 7.4
7.6 Advantages of Using Serum 7.6
7.7 Disadvantages of Serum Media 7.6
7.8 Defined Media 7.6
7.9 Function of Media Components 7.7
7.10 Suppliment of the Serum Free Media 7.9
7.11 Advantage of Serum Free Medium 7.10

4. 7.12 Disadvantages of Serum Free Medium
7.13 Some Commercial Serum Free Media
7.14 Artificial Serum
Brain Quest
8. Explant Isolation
8.1 Explant
8.2 Methods of Explant Isolation
8.3 Mechanical Isolation
8.4 Enzyme Desegregation
Brain Quest
9. Cell Counting and Viability Assay
9.1 Cells Viability Assay
9.2 Cell Proliferation Assay
9.3 Assay Techniques
9.4 Viability Stains
9.5 Cell Counting (Sorting) Tools
9.6 Direct Methods of Cell Counting
9.7 Biochemical Methods
9.8 Separation of Cells
9.9 Cell Separation based on Charge and Affinity
9.10 Metabolism of Toxicity
9.11 Assay System for Cytotoxicity
9.12 Proliferation Assay
9.13 Apoptosis Assay
9.14 Activation Assay
9.15 Cell Synchronization
9.16 Method of Cell Synchronization
Brain Quest
10. Growth Phases of Cell in Culture
10.1 Introduction
10.2 Growth Phases
10.3 Growth Response Curve
Brain Quest
11. Cell Differentiation
11.1 Differentiation
11.2 Classification of Differentiation
11.3 Parameters Governing the Entry of Cells into Differentiation
11.4 Fibronectin Helps in Cell Attachment
Brain Quest
Contents xi
7.10
7.11
7.12
7.13
8.1-8.5
8.1
8.1
8.1
8.3
5.5
9.1-9.20
9.1
9.2
9.2
9.3
9.5
9.7
9.10
9.10
9.11
9.13
9.13
9.13
9.15
9.17
9.18
9.18
9.19
10.1-10.5
10.1
10.1
10.4
70.5
11.1-11.4
11.1
11.1
11.2
11.3
11.4

5. xii Contents
12. Primary and Secondary Cell Culture 12.1-12.9
12.1 Introduction 12.1
12.2 Cell Culture Systems 12.1
12.3 Functional Characteristics 12.2
12.4 Primary Explantation Techniques 12.3
12.5 Subculturing 12.5
12.6 Process of Subculture 12.6
12.7 Serial Subculture, Split Ratio and Doubling Time 12.7
12.8 Applications of Cell Culture 12.7
Brain Quest 12.9
13. Techniques in Cloning of the Cells 13.1-13.7
13.1 Cloning 13.1
13.2 Methods of Cloning 13.1
13.3 Cloning of Non-adherent Cells 13.2
13.4 Quantitative Measures 13.2
13.5 Isolation of Clones 13.3
Brain Quest 13.7
14. Maintenance of the Cell Culture 14.1-14.14
14.1 Maintenance of the Cell 14.1
14.2 Subculturing Increases Life Span of Cells 14.1
14.3 Maintenance of the Adherent and Non-adherent Cells 14.2
14.4 Metabolic Capabilities of the Finite Cells 14.2
14.5 Growth Response Curve 14.3
14.6 Finite and Cancerous Cell Line 14.4
14.7 Characterization of Cells 14.9
14.8 Cross-contamination 14.10
14.9 Developments of Continuous Cell Line 14.11
14.10 Factor Affecting Gene Expression 14.13
14.11 Application of Cell Lines 14.14
Brain Quest 14.14
15. Cryopreservation of Animal Cells 15.1-15.5
15.1 Cryopreservation 15.1
' 15.2 Cryopreservation in Liquid Nitrogen 15.1
15.3 Methods of Preservation 15.2
15.4 Revival of Cells 15.3
15.5 Factors Favoring Good Survival 15.3
15.6 Banking of Cell Lines 15.3
15.7 Key to Successful Cryopreservation 15.4
15.8 Advanatages of Cryopreservation 15.4
Brain Quest 15.5

6. Contents xiii
16. Culture of Specific Cells 16.1-16.5
16.1 Culture of Specific Cells 16.1
16.2 Epithelial Cell Culture 16.1
16.3 Stem Cell Culture 16.2
16.4 Hematopoietic Cells ' 16.2
Brain Quest 16.5
17. Organ Culture 17.1-17.15
17.1 History and Development 17.1
17.2 Histotypic Culture 17.1
17.3 Organotypic Culture 17.4
17.4 Organ Culture 17.5
17.5 Techniques of Organ Culture 17.5
17.6 Advantage of Organ Culture 17.8
17.7 Limitations of The Organ Culture 17.8
17.8 Limiting Factors for 3D Organizations 17.8
17.9 Applications 17.8
17.10 Organotypic Culture 17.10
17.11 Tissue Engineering 17.11
17.13 Applications of Tissue engineering 17.14
Brain Quest 17.15
18. Cell to Cell Interactions 18.1-18.9
18.1 Role of Extracellular Matrix 18.1
18.2 Cell Junction 18.3
18.3 Gap Junction 18.3
18.4 Cell Adhesion Molecules (CAM) 18.4
18.5 Components of Extracellular Matrix 18.7
Brain Quest 18.8
19. Hybridoma Technology 19.1-19.22
19.1 Introduction 19.1
19.2 History 19.1
19.3 Cell Fusion Technology 19.2
19.4 Disadvantages of Conventional Antibodies 19.3
19.5 Advantages of Monoclonal Antibodies 19.3
19.6 Hybrid Cell Selection on HAT Medium 19.3
19.7 Types of Antibody 19.4
19.8 Nomenclature 19.7
19.9 Large Scale Production of Antibodies 19.8
19.10 Cloning - 19.10
19.11 Purification 19.13
19.12 Antibody Engineering 19.16

7. xiv Contents
19.13 Application of MAB 19.18
19.14 Advantages and Limitations 19.21
Brain Quest 19.22
20. Gene Therapy 20.1-20.29
20.1 Introduction 20.1
20.2 History of Gene Therapy 20.1
20.3 Types of Gene Therapy 20.3
20.4 Ex-Vivo and In Vivo Gene Therapy 20.3
20.5 Technical Aspects of Gene Therapy 20.4
20.6 Limitations 20.5
20.7 Gene-transfer Systems (Transfection Vector) 20.6
20.8 Viral Vectors 20.7
20.9 Pseudotyping 20.11
20.10 Non-viral Methods , ' 20.12
20.11 Hybrid Methods 20.16
20.12 Physical Methods 20.18
20.13 Comparison of Vectors 20.21
20.14 Role of Promoter 20.21
20.15 Regulated Gene Expression 20.22
20.16 Application and Advantages 20.23
20.17 Gene Therapy for Cancer Treatment 20.25
Brain Quest 20.29
21. Transgenic Animals 21.1-21.33
21.1 Introduction 21.1
21.2 Historical developments 21.1
21.3 Classification of Traits 21.1
21.4 Genetic Manupulation 21.2
21.5 Viral Mediated Gene Transfer 21.4
21.6 Animal Cloning 21.13
21.7 Nuclear Transfer Technology 21.16
21.8 Transgenic Mice 21.19
21.9 Transgenic Sheep 21.20
21.10 Method to Get Stable Transgenic Animal 21.21
21.11 Detection of Transgenic Animals 21.21
21.12 Gene Expression Regulation 21.26
21.13 Promoters 21.27
21.14 Special Vectors for Expression 21.30
21.15 Advantages and Limitations 21.31
21.16 Applications 21.31
Brain Quest 21.32

8. Contents xv
22. Application of Transgenic Animals 22.1-22.24
22.1 Historical 22.1
22.2 Introduction 22.1
22.3 Medicinal Applications 22.4
22.4 Proteins of Medical Importance 22.8
22.5 Supermouse 22.12
22.6 Antibody Production 22.12
22.7 Genetic Models 22.13
22.8 Agricultural Applications 22.14
22.9 Industrial Applications 22.17
22.10 Improvement in Fiber Production (Wool) 22.18
22.11 Growth and Carcass Composition 22.19
22.12 Enviro-pig 22.20
22.13 Xenotransplantation 22.20
22.14 Production of Hemoglobin 22.21
22.15 Transgenic Chickens 22.21
22.16 Immunocastration 22.22
22.17 The Future 22.22
22.18 Ethical Issues in Transgenic Animals 22.22
Brain Quest 22.24
23. Assisted Reproductive Technology (ART) 23.1-23.13
23.1 Introduction 23.1
23.2 Causes of Infertility 23.1
23.3 In-Vitro Fertilization (IVF) 23.2
23.4 Expansions of IVF 23.6
23.5 Surrogate Motherhood 23.9
23.6 Preimplantation Genetic Diagnosis (PGD) 23.9
23.7 Post-implantation Methods 23.10
23.8 Amniocentesis 23.10
23.9 Ectopic Pregnancy 23.10
23.10 Problems and Concerns 23.11
23.11 Terminology 23.12
Brain Quest 23.13
Index 1.1-1.7