Animal Cell Culture: Principles and Practice

Preface
Contents
Chapter 1: Overview to Animal Cell Culture
1.1 Introduction
1.2 Types of Cell Cultures
1.2.1 Primary Cell Culture
1.2.2 Secondary Cell Culture
1.2.3 Cell Line and Cell Strain
1.3 Ethical Considerations in Animal Tissue Culture
1.4 Common Nomenclatures in Animal Cell Culture
References
Chapter 2: Cell Culture Laboratory
2.1 Laboratory Design, Planning and Layout
2.1.1 Ventilation
2.1.2 Requirements
2.1.3 Services
2.1.4 Layout
2.1.4.1 Sterile Handling Area
2.1.4.2 Laminar Flow
2.1.4.3 Service Bench
2.1.4.4 Quarantine and Containment
2.1.4.5 Incubation
2.1.4.6 Preparation Area
2.1.4.7 Storage
2.2 Equipment and Materials for Cell Culture Laboratory
2.2.1 Aseptic Area
2.2.1.1 Laminar Flow Hood
2.2.1.2 Service Carts
2.2.1.3 Pipette Aids, Automatic Pipetting and Dispensing
2.2.1.4 Peristaltic Pump
2.2.1.5 Inverted Microscope
2.2.1.6 Centrifuge
2.2.1.7 Cell Counter
Haemocytometer Slide
Electronic Cell Counter
Cell Sizing
2.2.1.8 CCD Camera and Monitor
2.2.1.9 Dissecting Microscope
2.2.2 Incubation and Culture
2.2.2.1 Incubator
2.2.2.2 Humid CO2 Incubator
2.2.2.3 Water Bath
2.2.2.4 Roller Racks
2.2.2.5 Magnetic Stirrer
2.2.2.6 Temperature Recorder
2.2.2.7 Culture Vessels
2.2.3 Preparation and Sterilisation
2.2.3.1 Washup
Sinks or Soaking Baths
Glassware Washing Machine
Pipette Washer
Pipette Dryer
Drying Oven
2.2.3.2 Preparation of Media and Reagents
Water Purifier
Balances
Hot plate Magnetic Stirrer
pH Meter
Conductivity Meter
Osmometer
Bottling: Automatic Dispensers
2.2.3.3 Sterilisation
Sterilising Oven
Sterilisation Filters
Steam Steriliser (Autoclave)
2.2.3.4 Storage
Consumables
Refrigerators and Freezers
Cryostorage Containers
Controlled Rate Freezer
2.2.3.5 Supplementary Laboratory Equipment
Computers and Networks
Upright Microscope
Low-Temperature Freezer
Confocal Microscope
PCR Thermal Cycler
2.2.3.6 Specialised Equipment
Microinjection Facilities
Colony Counter
Centrifugal Elutriator
Flow Cytometer
Chapter 3: Good Laboratory Practices in Animal Cell Culture Laboratory and Biosafety Measures
3.1 Introduction
3.2 Biosafety Measures in Animal Cell Culture Laboratory
3.2.1 Biosafety Level 1 (BSL-1)
3.2.2 GLPs for Biosafety Level 1
3.2.3 Biosafety Level 2 (BSL-2)
3.2.4 GLPs for Biosafety Level 2
3.2.5 Biosafety Level 3
3.2.6 GLPs for Biosafety Level 3
3.2.7 Biosafety Level 4 (BSL-4)
3.2.8 GLPs for Biosafety Level 4
3.2.9 Safe Laboratory Practices
3.2.10 Common Good Lab Practices (GLPs) in Cell Culture
3.2.11 Waste Segregation
3.2.12 Types of Waste
3.2.13 Waste Management
References
Chapter 4: Managing Sterility in Animal Cell Culture Laboratory
4.1 Introduction
4.2 Elements of Aseptic Techniques
4.2.1 Sterile Work Area
4.2.2 Good Personal Hygiene
4.2.3 Media and Sterile Reagents
4.2.4 Sterile Handling
4.2.5 Use of Safety Cabinets
4.2.6 Culture Sterility
4.3 Biological Contamination
4.3.1 Bacteria
4.3.2 Yeasts
4.3.3 Moulds
4.3.4 Viruses
4.3.5 Mycoplasma
4.3.6 Cross-contamination
4.4 Aseptic Technique Checklist
References
Chapter 5: Media and Buffer Preparation for Cell Culture
5.1 Introduction
5.2 Media
5.2.1 Physiochemical Properties of Media
5.2.2 Components of Media
5.2.3 Types of Culture Medium
5.3 Methodology to Prepare Cell Culture Media
5.3.1 Preparation of Incomplete DMEM Media
References
Chapter 6: Properties of Cultured Cells and Selection of Culture Media
6.1 Introduction
6.2 Systems for Growing Cell Culture
6.3 Morphological Differences in Mammalian Cell
6.4 Maintaining Cultured Cells
6.5 When to Subculture
6.5.1 Cell Density
6.5.2 Exhaustion of Medium
6.5.3 Subculture Schedule
6.6 Media Recommendations
6.6.1 Culture Media
6.6.2 Serum
6.6.3 pH
6.6.4 CO2
6.6.5 Temperature
6.6.6 Different Culture Mediums for Different Cells
References
Chapter 7: Selection and Maintenance of Cultured Cells
7.1 Introduction
7.2 Origin and Characterisation of Cells
7.2.1 Cell Differentiation
7.3 Selection of Appropriate Cell Line
7.3.1 Techniques for Detachment of Cells
7.3.2 Source of Tissue
7.3.3 Subculture
7.3.4 Growth Conditions and Characteristics
7.3.5 Other Criteria
7.4 Maintenance of Cell Line
7.5 Media
7.6 Conclusion
References
Chapter 8: Inoculation and Passaging of Adherent and Suspension Cells
8.1 Introduction
8.2 Subculturing in Monolayer Cultures
8.3 Troubleshooting Monolayer Cell Subculturing
8.3.1 Dissociation of Cells
8.3.2 Cell Clump Formation Post-dissociation
8.3.3 Cells Reattachment Issues
8.3.4 Reduced Viability
8.4 Subculturing in Suspension Cell Cultures
8.5 Media Consumption by Cell Lines
8.6 Types of Cell Detachment Techniques
8.6.1 Degree of Cell Adhesion
8.6.2 Use of Detached Cells
8.6.3 Process Compatibility
8.6.4 Culture Support
8.6.5 Process Scale
8.6.6 Regulatory Constraints
8.6.7 Temporal Resolution
8.6.8 Compatibility with Sterilisation Methods
8.6.9 Production Costs
8.7 Different Cell Detachment Methods
8.8 Role of Cellular Density in the Subculturing Procedures
8.9 Determining the Time Interval and Schedule in Subculturing Process
8.10 Cellular Synchronisation
8.10.1 Centrifugal Elutriation
8.10.2 Chemical Blockade
8.10.3 Arrest in the City of M
8.10.4 Nocodazole
8.10.5 Inhibition of CDK1
8.10.6 Colchicine
8.10.7 Arrest in S (Arrest in G1/S)
8.10.8 Double Thymidine Block
8.10.9 Other Methods of Cell Synchronisation
8.10.9.1 Mitotic Selection
8.10.9.2 Nutrient/Serum Deprivation
8.10.9.3 Contact Inhibition
8.11 Cellular Interaction and Signalling Mechanism
8.12 Attaining Standard Growth Cycles and Split Ratios
8.13 Using Antibiotics for Contamination Control
8.14 Conclusion
References
Chapter 9: Counting of Cells
9.1 Introduction
9.2 Cell Growth, Cell Propagation, and Cell Viability
9.3 Earlier Methods of Cell Counting
9.3.1 Recent Techniques in Cell Counting
9.4 Direct Cell Counting
9.5 Indirect Cell Counting
9.6 Errors in Cell Counting
9.7 Conclusion
References
Chapter 10: Cryopreservation of Cell Lines
10.1 Introduction
10.2 Fundamentals of Cryopreservation
10.3 Cryoprotective Agents
10.4 Polymers
10.5 Glycerol
10.6 Dimethyl Sulphoxide (DMSO)
10.7 Proteins
10.8 Primary Mechanism of Action of Cryoprotectants
10.9 The Cell Banker Series
10.10 Cryoinjury
10.11 Varied Application of Cryopreservation Technique on Special Cell Cultures/Cells
10.12 Female Germ Cells (Oocytes and Embryos)
10.13 Male Germ Cells (Sperm, Testicular Tissue and Semen)
10.14 Hepatocytes
10.15 Stem Cells
10.16 Other Frequently Cryopreserved Cells
10.17 Optimum Cooling Rate
10.18 Influence of Warming Rate
10.19 Limitation of Cryopreservation
10.20 Vitrification
10.21 Conclusion
References
Chapter 11: Resuscitation of Frozen Cell Lines
11.1 Introduction
11.2 Freezing Cell Lines (Cryopreservation)
11.3 Thawing Cell Lines
11.4 Cell Thawing Techniques
11.4.1 Hand-Warming
11.4.2 Water Bath
11.4.3 Specialised Devices
11.4.4 Bead Bath
11.5 Material and Reagents Employed for Resuscitation
11.5.1 Materials
11.5.2 Reagents and Solvents
11.6 Methodology for Cell Revival
11.6.1 Equipment Setup
11.6.2 Thawing Protocol
11.7 Critical Parameters for Resuscitation
11.8 Conclusion
References
Chapter 12: Isolation and Culturing of Cells from Different Tissues
12.1 Introduction
12.2 General Methods to Isolate Cells from Tissues
12.2.1 Mechanical Dissociation
12.2.2 Enzymatic Dissociation
12.2.2.1 Disaggregation by Trypsin
12.2.2.2 Disaggregation by Collagenase
12.2.2.3 Other Enzymes
12.2.3 Primary Explant Technique
12.2.4 Chemical Dissociation
12.3 Segregation of Viable and Non-viable Cells
12.4 Summary
References
Chapter 13: Co-culture Techniques
13.1 Introduction
13.2 Co-cultures
13.3 Extracellular Microenvironment
13.4 Variables in Co-cultures
13.4.1 Cell Communication During Co-cultures
13.4.2 Cell Culture Medium
13.4.3 Cell Population in Co-cultures
13.4.4 Volume of Co-cultures
13.5 Model Systems in Co-cultures
13.5.1 Computational Models
13.5.2 In Vitro Tissue Models
13.5.2.1 Skin
13.5.2.2 Liver
13.5.2.3 Blood Vessels
13.5.2.4 Blood Brain Barrier
13.5.2.5 Bone
13.6 Potential Challenges with Co-cultures
13.7 Future Perspective
13.8 Conclusion
References
Chapter 14: 3D Cell Culture Techniques
14.1 Introduction
14.2 3D Cell Culture Versus 2D Cell Culture Systems
14.3 Overview of 3D Cell Culture Techniques
14.4 Methods of 3D Cell Culture Techniques
14.4.1 Scaffold-Based Techniques
14.4.2 Scaffold-Free Techniques
14.5 Stem Cells in 3D Spheroids and Organoids
14.6 Applications of 3D Cell Culture Techniques
14.7 Challenges in 3D Culture Techniques
14.8 Conclusion
14.9 Future Prospects
References
Chapter 15: Stem Cell Culture Techniques
15.1 Introduction
15.2 Historical Background
15.3 Properties of Stem Cells
15.4 Sources of Stem Cells
15.4.1 Human Umbilical Cord
15.4.2 Bone Marrow
15.4.3 Adipose Tissue
15.4.4 Amniotic Fluid
15.4.5 Dental Pulp
15.5 Classification of Stem Cell Types Based on Origin
15.5.1 Embryonic Stem Cells
15.5.2 Adult Stem Cells
15.5.3 Pluripotent Stem Cells
15.6 Classification of Stem Cells Based on Potency
15.6.1 Totipotent
15.6.2 Pluripotent
15.6.3 Multipotent
15.6.4 Oligopotent
15.6.5 Unipotent
15.7 Stem Cell Culture Techniques
15.7.1 Stem Cell Lines
15.7.2 Standardisations for Specific Uses
15.7.3 Maintenance of Aseptic Condition
15.7.4 Safety Features for Stem Cell Culturing
15.7.5 Technique of Embryonic Stem Cell Culture (Fig. 15.3)
15.7.6 Thawing of Frozen Stocks
15.7.7 Gelatin Coating of Culture Plates
15.7.8 Media Change
15.7.9 Passaging
15.7.10 Cryopreservation
15.7.11 Precautions and Troubleshooting Tips (Table 15.3)
15.8 Limtations and Challenges in Stem Cell Research
15.9 Applications of Stem Cells
15.10 Conclusion
15.11 Future Prospective
References
Chapter 16: Identification and Removal of Biological Contamination in the Media and Cell Suspensions
16.1 Introduction
16.2 Cell Culture Contaminants
16.2.1 Biological Contaminants
16.2.1.1 Mycoplasma
16.2.1.2 Bacteria
16.2.1.3 Fungi
16.2.1.4 Virus
16.2.1.5 Yeast
16.2.2 Chemical Contaminants
16.2.3 Inorganic Ions
16.2.4 Organic Compounds
16.3 Role of Antibiotics and Antimycotics
16.4 Sources of Contamination
16.4.1 Biological Sources
16.4.2 Chemical Sources
16.4.2.1 Water
16.4.2.2 Sera
16.5 Testing and Detection of Different Contaminants in Media
16.6 Testing and Detection of Different Contaminants in Cell Cultures
16.6.1 Bacteria
16.6.2 Fungi
16.6.3 Virus
16.6.4 Mycoplasma
16.6.5 Yeast
16.7 Problem of Cross-Contamination by Other Species
16.7.1 Methods for the Identification and Elimination of Cross-Contamination
16.8 Effects of Contamination on Media and Cell Suspensions
16.9 Prevention of Possible Contamination and Safe Handling of Cell Cultures – Maintaining Aseptic Conditions
16.10 Conclusion
References
Chapter 17: Analysis of Cell Growth Kinetics in Suspension and Adherent Types of Cell Lines
17.1 Introduction
17.2 Kinetic Characterisation of Cell Culture
17.3 Cell Kinetics in Batch Culture
17.3.1 Lag Phase
17.3.2 Log Phase
17.3.3 Time Profile of the Concentration of Cells, Nutrients and Metabolites
17.3.4 Variation of Cell Morphology
17.3.5 Determination of Cell Population Heterogeneity for Kinetic Studies
17.3.6 Cell Kinetics in Continuous Culture
17.3.7 Advantages of Continuous Culture
17.3.8 Applications of Continuous Culture
17.3.8.1 Estimation of Specific Rates of Cell Metabolism in Continuous Culture
17.4 Influences of Physiological Conditions and Rate Equations
17.4.1 Effect of Nutrients
17.4.2 Effect of Temperature
17.4.3 Effect of pH
17.4.4 Dissolved Oxygen and Oxygen Uptake Rate
17.4.5 Dissolved Carbon Dioxide
17.4.6 Osmolality and Salts
17.4.7 The Rate Law for Cell Growth, Death and Productivity
17.5 Conclusion
References
Chapter 18: In Vitro Cytotoxicity Analysis: MTT/XTT, Trypan Blue Exclusion
18.1 Introduction
18.2 Categories of In Vitro Cytotoxicity and Cell Viability Assays
18.2.1 Dye Exclusion Methods
18.2.2 Colorimetric Methods
18.2.3 Fluorometric and Luminometric Assays
18.3 Commonly Used In Vitro Cytotoxicity Analysis Methods
18.3.1 Trypan Blue Exclusion (TBE)
18.3.1.1 Materials
18.3.1.2 Protocol
18.3.2 MTT Assay
18.3.2.1 Materials
18.3.2.2 Protocol
18.3.3 XTT Assay
18.3.3.1 Materials
18.3.3.2 Protocol
18.3.4 Sulforhodamine B (SRB) Assay
18.3.4.1 Materials
18.3.4.2 Procedure
18.3.5 LDH Assay
18.3.5.1 Materials/Reagents
18.3.5.2 Procedure
References
Chapter 19: Applications of Animal Cell Culture-Based Assays
19.1 Introduction
19.2 Biomarker Identification
19.2.1 Cell Culture-Based Cancer Biomarker Identification
19.3 Genetic Manipulation
19.3.1 General Scheme for Genetic Engineering of Animal Cells in Culture for Protein Production
19.3.2 Applications of Genetic Manipulation of Animal Cells in Culture
19.3.2.1 Recombinant Therapeutic Protein Production
19.3.2.2 Gene Therapy
19.3.3 Advanced Editing Tools
19.4 Pathological Studies
19.5 Pharmaceutical Studies
19.5.1 Drug Screening in Cell Lines
19.6 Stem Cell Research
19.7 Cellular Development and Differentiation
19.8 Hybridoma Technology
References
Chapter 20: Ethical Issues in Animal Cell Culture
20.1 Introduction
20.2 Ethical Concerns in Handling Animal Cell Culturing
20.2.1 Acquisition of Cell Culture
20.2.2 Authentication of Cell Culture
20.2.3 Characterization of Cell Culture
20.2.4 Isolation of Cell Lines
20.3 Ethical Concerns Associated with Chimera Effect in Tissue Engineering
20.4 Ethics Related to Development and Preservation (Cryopreservation) of Cell Cultures
20.5 Ethical Conduct in Cell Culture Handling
20.5.1 Maintaining the Instability
20.5.2 Contamination and Non-specific Identification
20.5.3 Transfer of Cell Line Between Laboratories
20.5.4 Use of Equipment and Media
20.6 Dilemmas in Tissue Engineering and Tissue Banking
20.7 Cell Culturing Associated with Intellectual Property and Legal Rights
20.7.1 Laws Concerning Intellectual Property
20.8 Public and Scientific Community Opinions on Cell Culturing
20.9 Conclusion
References
Chapter 21: Common Troubleshooting Methods in Cell Culture Techniques
21.1 Introduction
21.2 Troubleshooting
21.2.1 Why Do Sometimes Cells Not Stay Viable After Thawing the Stock?
21.2.2 Why Do Cells Seem to Grow Slowly Sometimes?
21.2.3 What Is the Troubleshooting in Cell Counting?
21.2.4 What Causes Cell Clumping?
21.2.5 What Leads to a Cross-Linked or Misidentified Cell Line?
21.2.6 Why Do Sometimes Cells Do Not Revive After Being Cryopreserved?
21.2.7 What Are the Sources of Contamination and How Should They Be Rectified?
21.2.8 What Causes Unexpected Cell Detachment?
21.2.9 What Is the Troubleshooting in Cytotoxicity Assay?
21.2.10 What Triggers a Rapid pH Shift in the Medium?
21.2.11 What Leads to Precipitation in the Medium and Either Causes a Change in pH or No Change in pH?
21.3 Conclusion
References
Index