Animal Cell Culture: Principles and Practice

Description

This introductory guide provides novice researchers and lab students with a thorough step-by-step approach to standard animal cell culture techniques. Coverage includes lab safety and best practices, sterility management, preparation, ethical considerations, and troubleshooting for common pain points. This is an up-to-date, indispensable handbook for early-career researchers and students, as well as established scientists in biotechnology, cell and developmental biology, pharmaceutical toxicology, cytogenetics, and more.  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