Freshney’s Culture of Animal Cells
, by Freshney, R. Ian; Capes-Davis, Amanda; Gregory, Carl; Przyborski, Stefan
- ISBN: 9781119513018 | 1119513014
- Cover: Cloth
- Copyright: 12/10/2020
FRESHNEY’S CULTURE OF ANIMAL CELLS Provides comprehensive single-volume coverage of basic skills and protocols, specialized techniques and applications, and new and emerging developments in the field Covers every essential area of animal cell culture, including lab design, disaster and contingency planning, safety, bioethics, media preparation, primary culture, mycoplasma and authentication testing, cell line characterization and cryopreservation, training, and troubleshooting Features a wealth of new content including protocols for gene delivery, iPSC generation and culture, and tumor spheroid formation Includes an updated and expanded companion website containing figures, artwork, and supplementary protocols to download and print
THE NEW EDITION OF THE LEADING TEXT ON THE BASIC METHODOLOGY OF CELL CULTURE, FULLY UPDATED TO REFLECT NEW APPLICATIONS INCLUDING IPSCS, CRISPR, AND ORGAN-ON-CHIP TECHNOLOGIES
Freshney’s Culture of Animal Cells is the most comprehensive and up-to-date resource on the principles, techniques, equipment, and applications in the field of cell and tissue culture. Explaining both how to do tissue culture and why a technique is done in a particular way, this classic text covers the biology of cultured cells, how to select media and substrates, regulatory requirements, laboratory protocols, aseptic technique, experimental manipulation of animal cells, and much more.
The eighth edition contains extensively revised material that reflects the latest techniques and emerging applications in cell culture, such as the use of CRISPR/Cas9 for gene editing and the adoption of chemically defined conditions for stem cell culture. A brand-new chapter examines the origin and evolution of cell lines, joined by a dedicated chapter on irreproducible research, its causes, and the importance of reproducibility and good cell culture practice. Throughout the book, updated chapters and protocols cover topics including live-cell imaging, 3D culture, scale-up and automation, microfluidics, high-throughput screening, and toxicity testing. This landmark text:
The eighth edition of Freshney’s Culture of Animal Cells is an indispensable volume for anyone involved in the field, including undergraduate and graduate students, clinical and biopharmaceutical researchers, bioengineers, academic research scientists, and managers, technicians, and trainees working in cell biology, molecular biology, and genetics laboratories.
AMANDA CAPES-DAVIS, PHD, is a cell culture scientist and technical writer. She was Founding Manager and Honorary Scientist at CellBank Australia, Children’s Medical Research Institute (CMRI), and is a member of the International Cell Line Authentication Committee (ICLAC). She was a Reviewing Editor for the 7th edition of Culture of Animal Cells, and has written numerous journal articles, policies, protocols, and white papers on good cell culture practice.
R. IAN FRESHNEY, PHD, was an honorary Senior Research Fellow at the Institute of Cancer Sciences at the University of Glasgow, UK. Dr Freshney, who died in 2019, was a world-renowned cancer biologist and a pioneer in cell culture techniques who made important contributions to new approaches for treating cancer patients. He taught cell culture courses at national and international level, and wrote and edited numerous books, including the first seven editions of Culture of Animal Cells.
List of Figures
List of Color Plates
List of Tables
Foreword
Acknowledgements
Abbreviations
Book Navigation
I UNDERSTANDING CELL CULTURE
1 Introduction
1.1 Terminology
1.1.1 Tissue Culture and Cell Culture
1.1.2 Sources of Terminology
1.2 Historical Development
1.2.1 Substrates and Media
1.2.2 Primary Cultures and Cell Lines
1.2.3 Organ, Organotypic, and Organoid Culture
1.3 Applications
1.4 Advantages of Tissue Culture
1.4.1 Environmental Control
1.4.2 Homogeneity and Characterization
1.4.3 Economy, Scale, and Automation
1.4.4 Replacement of In Vivo Models
1.5 Limitations of Tissue Culture
1.5.1 Quality and Expertise
1.5.2 Quantity and Cost
1.5.3 Limited Species and Cell Types
1.5.4 Limited Understanding of the Cell and its Microenvironment
2 Biology of Cultured Cells
2.1 The Culture Environment
2.2 Cell Adhesion
2.2.1 Intercellular Junctions
2.2.2 Cell Adhesion Molecules
2.2.3 Cytoskeleton
2.2.4 Extracellular Matrix (ECM)
2.2.5 Cell Motility
2.3 Cell Division
2.3.1 Cell Cycle
2.3.2 Control of the Cell Cycle
2.4 Cell Fate
2.4.1 Embryonic Lineages
2.4.2 Stem Cells and Potency
2.4.3 Differentiation
2.4.4 Control of Potency and Differentiation
2.4.5 Lineage Commitment
2.4.6 Lineage Plasticity
2.5 Cell Death
3 Origin and Evolution of Cultured Cells
3.1 Origin of Cultured Cells
3.1.1 Sample Origin
3.1.2 Disease Origin
3.2 Evolution of Cell Lines
3.2.1 Phases of Cell Cultivation
3.2.2 Clonal Evolution
3.3 Changes in Genotype
3.3.1 Chromosomal Aberrations
3.3.2 Genomic Variation
3.4 Changes in Phenotype
3.4.1 Phenotypic Variation
3.4.2 Phenotype and Culture Conditions
3.5 Senescence and Immortalization
M3.1 Senescence and Immortalization
3.5.1 Intrinsic Control of Senescence
3.5.2 Extrinsic Control of Senescence
3.6 Transformation
3.6.1 Characteristics of Transformation
3.6.2 Aberrant Growth Control
3.6.3 Tumorigenicity and Malignancy
3.7 Conclusions: Origin and Evolution
II LABORATORY AND REGULATORY REQUIREMENTS
4 Laboratory Design and Layout
4.1 Design Requirements
4.1.1 General Design Considerations
4.1.2 User Requirements
4.1.3 Regulatory Requirements
4.1.4 Engineering Requirements
4.2 Layout of Laboratory Areas
4.2.1 Sterile Handling Area
4.2.2 Incubation Area
4.2.3 Quarantine Area
4.2.4 Preparation Area
4.2.5 Washup Area
4.2.6 Storage Area
4.3 Disaster and Contingency Planning
4.3.1 Contingency Plans and Priorities
4.3.2 Equipment Monitoring and Alarms
S4.1 Designing a Warmroom
5 Equipment and Materials
5.1 Sterile Handling Area Equipment
5.1.1 Biological Safety Cabinet (BSC)
5.1.2 BSC Services and Consumables
5.1.3 Sterile Liquid Handling Equipment
5.1.4 Centrifuge
5.2 Imaging and Analysis Equipment
5.2.1 Microscopes
5.2.2 Cameras
5.2.3 Computer and Monitor
5.2.4 Cell Counting and Analysis Equipment
5.3 Incubation Equipment
5.3.1 Incubators
5.3.2 Incubator Accessories
5.3.3 Water Baths
5.4 Preparation and Washup Equipment
5.4.1 Water Purification Systems
5.4.2 Preparation Equipment
5.4.3 Washup Equipment
5.4.4 Sterilization Equipment
5.5 Cold Storage Equipment
5.5.1 Refrigerators and Freezers
5.5.2 Cryofreezers
5.5.3 Rate-Controlled Freezer
6 Safety and Bioethics
6.1 Laboratory Safety
6.1.1 Risk Assessment
6.1.2 Safety Regulations
6.1.3 Training
6.1.4 Ergonomics
6.2 Hazards in Tissue Culture Laboratories
6.2.1 Needlestick and Sharps Injuries
6.2.2 Hazardous Substances
6.2.3 Asphyxia and Explosion
6.2.4 Burns and Frostbite
6.2.5 Fire
6.2.6 Equipment Hazards
6.3 Biosafety
6.3.1 Source of Biohazard Risk
6.3.2 Biohazard Risk Groups
6.3.3 Biological Containment Levels
6.3.4 Containment Equipment
6.3.5 Decontamination and Fumigation
6.3.6 Waste Disposal and Disinfectants
6.3.7 Genetically Modified Organisms (GMOs)
6.4 Bioethics
6.4.1 Ethical Use of Animal Tissue
6.4.2 Ethical Use of Human Tissue
6.4.3 Donor Consent
S6.1 Hierarchy of Risk Controls
S6.2 Ionizing Radiation
S6.3 Biosecurity
S6.4 Donor Privacy
7 Reproducibility and Good Cell Culture Practice
7.1 Reproducibility
7.1.1 Terminology: Reproducible Research
7.1.2 Causes of Irreproducible Research
7.1.3 Solutions to Irreproducible Research
7.2 Good Practice Requirements
7.2.1 Good Cell Culture Practice (GCCP)
7.2.2 Good Laboratory Practice (GLP)
7.2.3 Good Manufacturing Practice (GMP)
7.3 Cell Line Provenance
7.3.1 Provenance Information
7.3.2 Reporting for Publication
7.4 Validation Testing
7.4.1 Testing for Microbial Contamination
7.4.2 Testing for Authenticity
7.5 Quality Assurance
7.5.1 Standard Operating Procedures (SOPs)
7.5.2 Media and Reagents
7.5.3 Culture Vessels
7.5.4 Equipment
7.5.5 Facilities
7.6 Replicate Sampling
7.6.1 Experimental Design
7.6.2 Samples and Data
III MEDIUM AND SUBSTRATE REQUIREMENTS
8 Culture Vessels and Substrates
8.1 Attachment and Growth Requirements
8.2 Substrate Materials
8.2.1 Common Substrate Materials
8.2.2 Alternative Substrate Materials
8.3 Substrate Treatments
8.3.1 Substrate Conditioning
8.3.2 Extracellular Matrix (ECM) Coatings
P8.1 Application of Matrigel Coatings
8.3.3 Collagen and Gelatin
8.3.4 ECM Mimetic Treatments
8.3.5 Polymer Coatings
8.3.6 Nonadhesive Substrates and Patterning
8.3.7 Other Surface Treatments
8.4 Feeder Layers
8.5 Choice of Culture Vessel
8.5.1 Cell Yield
8.5.2 Multiwell Plates
8.5.3 Flasks and Petri Dishes
8.5.4 Multilayer Flasks
8.5.5 Lids and Venting
8.5.6 Uneven Growth
8.5.7 Cost
8.6 Application-specific Vessels
8.6.1 Imaging
8.6.2 Suspension Culture
8.6.3 Scaffold-free 3D Culture
8.6.4 Permeable Supports
8.6.5 Scaffold-based 3D Culture
9 Defined Media and Supplements
9.1 Medium Development
9.2 Physicochemical Properties
9.2.1 pH
9.2.2 Buffering
9.2.3 Carbon dioxide (CO2) and Sodium Bicarbonate (NaHCO3)
9.2.4 Oxygen
M9.1 Hypoxic Cell Culture
9.2.5 Temperature
9.2.6 Osmolality
9.2.7 Viscosity
9.2.8 Surface Tension and Foaming
9.3 Balanced Salt Solutions
9.4 Media Formulations
9.4.1 Amino Acids
9.4.2 Vitamins
9.4.3 Inorganic Salts
9.4.4 Glucose
9.4.5 Other Components
9.5 Serum
9.5.1 Protein
9.5.2 Hormones and Growth Factors
9.5.3 Nutrients and Metabolites
9.5.4 Lipids
9.5.5 Trace Elements
9.5.6 Inhibitors
9.6 Other Media Supplements
9.6.1 Conditioned Medium
9.6.2 Antibiotics
9.7 Choice of Complete Medium
9.7.1 Serum Testing
9.7.2 Serum Batch Reservation
9.7.3 Serum Treatment
9.8 Storage of Medium and Serum
S9.1 Preparation of pH standards
10 Serum-free Media
10.1 Rationale for Serum-free Medium
10.1.1 Disadvantages of Serum
10.1.2 Advantages of Serum-free Media
10.1.3 Disadvantages of Serum-free Media
10.2 Development of Serum-free Medium
10.3 Serum-free Media Formulations
10.4 Serum-free Supplements
10.4.1 Hormones and Growth Factors
10.4.2 Antioxidants, Vitamins, and Lipids
10.4.3 Other Supplements for Serum-free Medium
10.5 Serum Replacements
10.6 Use of Serum-free Medium
10.6.1 Choice of Serum-free Media
10.6.2 Preparation of Serum-free Media
10.6.3 Adaptation to Serum-free Media
10.7 Xeno-free Media
10.8 Animal Product-free Media
10.9 Conclusions: Serum-free Media
11 Preparation and Sterilization
11.1 Terminology: Preparation
11.2 Sterilization Methods
11.2.1 Dry Heat Sterilization
11.2.2 Pressurized Steam Sterilization (Autoclaving)
11.2.3 Irradiation
11.2.4 Plasma Sterilization
11.2.5 Chemical Sterilization
11.2.6 Filter Sterilization
11.2.7 Sterility Indicators
11.3 Glassware
P11.1 Preparation and Sterilization of Glassware
11.3.1 Detergent Selection
11.3.2 Glassware Sterilization
11.3.3 Caps and Closures
P11.2 Preparation and Sterilization of Screw Caps
11.4 Other Laboratory Apparatus
11.4.1 Cleaning and Packaging
11.4.2 Sterilization or Disinfection
11.5 Water
11.5.1 Water Purity
11.5.2 Purification Methods
P11.3 Preparation and Sterilization of Ultrapure Water (UPW)
11.5.3 Monitoring and Maintenance
11.6 Media and Other Reagents
11.6.1 Balanced Salt Solutions
P11.4 Preparation and Sterilization of DPBS-A
11.6.2 Basal and Complete Media
P11.5 Preparation of Medium from Powder
P11.6 Preparation of Medium from 10X Concentrate
P11.7 Preparation of Medium from 1X Stock
11.6.3 Serum
11.7 Sterile Filtration
11.7.1 Filter Selection
11.7.2 Disposable Filters
P11.8 Sterile Filtration with Syringe-tip Filter
P11.9 Sterile Filtration with Vacuum Filter Unit
11.7.3 Reusable Filter Assemblies
11.7.4 Filter Testing
11.8 Medium Testing
11.8.1 Sterility Testing
11.8.2 Culture Testing
S11.1 Preparation, Sterilization, and Use of Glass Pipettes
S11.2 Sterilization of Reusable Filter Assemblies
S11.3 Collection and Sterilization of Serum
S11.4 Sterile Filtration using Peristaltic Pump
S11.5 Sterile Filtration with Large In-line Filter
S11.6 Sterility Testing using Microbiological Culture
S11.7 Clonogenic Assay for Testing Medium
S11.8 Growth Curve Analysis for Testing Medium
IV HANDLING CULTURES
12 Aseptic Technique
12.1 Objectives of Aseptic Technique
12.1.1 Managing Contamination Risk
12.1.2 Maintaining Sterility
12.2 Elements of Aseptic Environment
12.2.1 Quiet Area
12.2.2 Laminar Airflow
12.2.3 Work Surface
12.2.4 Personal Protective Equipment (PPE)
12.2.5 Reagents and Media
12.2.6 Cultures
12.3 Sterile Handling
12.3.1 Swabbing
12.3.2 Flaming
12.3.3 Capping
12.3.4 Handling Bottles and Flasks
12.3.5 Pouring
12.3.6 Pipetting
12.3.7 Small-volume Dispensing
12.3.8 Large-volume Dispensing
12.4 Good Aseptic Technique
12.4.1 Aseptic Technique using Laminar Airflow
P12.1 Aseptic Technique Handling Flasks in a BSC
P12.2 Aseptic Technique Handling Dishes or Plates
12.4.2 Aseptic Technique on the Open Bench
P12.3 Working on the Open Bench
12.5 Controlling Equipment Contamination
12.5.1 Incubators
P12.4 Cleaning CO2 Incubators
12.5.2 Boxed Cultures
12.5.3 Gassing Cultures
13 Primary Culture
13.1 Rationale for Primary Culture
13.2 Initiation of Primary Culture
13.2.1 Proteases Used in Disaggregation
13.2.2 Other Agents Used in Disaggregation
13.2.3 Common Features of Disaggregation
13.3 Tissue Acquisition and Isolation
13.3.1 Nonhuman Tissue Samples
13.3.2 Mouse Embryo
P13.1 Isolation of Mouse Embryos
13.3.3 Chick Embryo
13.3.4 Human Biopsy Samples
P13.2 Handling Human Biopsies
13.4 Primary Explantation
P13.3 Culture of Primary Explants
13.5 Enzymatic Disaggregation
13.5.1 Trypsin
P13.4 Warm Trypsin Disaggregation
13.5.2 Trypsin with Cold Preexposure
P13.5 Cold Trypsin Disaggregation
13.5.3 Other Enzymatic Procedures
P13.6 Collagenase Disaggregation
13.6 Mechanical Disaggregation
P13.7 Mechanical Disaggregation by Sieving
13.7 Enrichment of Viable Cells
P13.8 Enrichment of Viable Cells
13.8 Record Keeping for Primary Culture
13.9 Conclusions: Primary Culture
S13.1 Isolation of Chick Embryos
S13.2 Disaggregation of Chick Embryo Organ Rudiments
S13.3 Maximal Serial Transfer (MST) of Human Fibroblasts from Skin Explants
14 Subculture and Cell Lines
14.1 Terminology: Cell Line and Subculture
14.2 Initiating a Cell Line
14.2.1 Cell Line Names and Identifiers
14.2.2 Culture Age
14.2.3 Cell Line Validation
14.3 Choosing a Cell Line
14.4 Maintaining a Cell Line
14.4.1 Routine Observation
14.4.2 Standardization of Culture Conditions
14.4.3 Use of Antibiotics
14.5 Replacing Medium (Feeding)
14.5.1 Criteria for Replacing Medium
14.5.2 Holding Medium
14.5.3 Standard Procedure for Feeding
P14.1 Feeding Adherent Cultures
14.6 Subculture (Passaging)
14.6.1 Criteria for Subculture
14.6.2 Dissociation Agents
14.6.3 Standard Procedure for Subculture
P14.2 Trypsinization of Adherent Cells
14.6.4 Growth Cycle and Split Ratios
14.7 Maintaining Suspension Cultures
14.7.1 Standard Procedure for Suspension Culture
P14.3 Subculture of Suspension Cells
14.8 Serum-free Subculture
14.9 Record Keeping for Cell Lines
15 Cryopreservation and Banking
15.1 Principles of Cryopreservation
15.1.1 Cryoprotectants
15.1.2 Cooling Rate
15.1.3 Storage Temperature
15.1.4 Vitrification
15.2 Apparatus for Cryopreservation
15.2.1 Cryovials
15.2.2 Controlled Cooling Devices
15.2.3 Cryofreezers
15.3 Requirements for Cryopreservation
15.3.1 When to freeze
15.3.2 Freezing Medium
15.3.3 Cell Concentration
15.4 Cryopreservation Procedures
15.4.1 Cryopreservation in Cryovials
P15.1 Freezing Cells in Cryovials
15.4.2 Cryopreservation in Other Vessels
15.4.3 Thawing Stored Cryovials
P15.2 Thawing Frozen Cryovials
15.4.4 Viability Testing
15.5 Cell Banking Procedures
15.5.1 Rationale for Cell Banking
15.5.2 Principles of Cell Banking
15.5.3 Replacement of Culture Stocks
15.6 Cell Repositories
15.7 Record Keeping for Frozen Stocks
15.8 Transporting Cells
S15.1 Shipping Cells
V VALIDATION AND CHARACTERIZATION
16 Microbial Contamination
16.1 Sources of Contamination
16.1.1 Operator Problems
16.1.2 Environmental Problems
16.1.3 Equipment Problems
16.1.4 Reagent Problems
16.1.5 Cell Line Problems
16.2 Management of Contamination
P16.1 Disposal of Contaminated Cultures
16.3 Visible Microbial Contamination
16.3.1 Testing of Bacteria, Fungi, and Yeasts
16.3.2 Eradication of Bacteria, Fungi, and Yeasts
P16.2 Treatment of Microbial Contamination
16.4 Mycoplasma Contamination
16.4.1 Mycoplasma Detection
P16.3 Detection of Mycoplasma by PCR
P16.4 Detection of Mycoplasma using Hoechst 33258
16.4.2 Mycoplasma Eradication
P16.5 Eradication of Mycoplasma Contamination
16.5 Viral Contamination
16.5.1 Detection of Viral Contamination
16.5.2 Eradication of Viral Contamination
16.6 Dealing with Persistent Contamination
17 Cell Line Misidentification and Authentication
17.1 Terminology: Cross-contamination, Misidentification, and Authentication
17.2 Misidentified Cell Lines
17.2.1 Impact
17.2.2 Causes
17.2.3 Eradication
17.3 Cell Line Authentication
17.3.1 Evolution of Authentication Techniques
17.3.2 Short Tandem Repeat (STR) Profiling
17.3.3 CO1 DNA Barcoding
P17.1 CO1 Barcoding of Animal Cells
17.3.4 Cytogenetic Analysis
P17.2 Chromosome Preparation and Giemsa Staining
17.4 Authentication of Challenging Samples
17.4.1 Cell Line Mixtures
17.4.2 Cell Lines with Microsatellite Instability (MSI)
17.4.3 Hybrid Cell Lines
17.5 Conclusions: Authentication
18 Cell Line Characterization
18.1 Priorities and Essential Characterization
18.1.1 Validation Testing
18.1.2 Morphology
18.1.3 Growth Curve Analysis
18.1.4 Transformation Assays
18.2 Genotype-based Characterization
18.2.1 Sequence Analysis
18.2.2 Cytogenetic Analysis
18.2.3 Epigenetic Analysis
18.3 Phenotype-based Characterization
18.3.1 Cell Line-specific Markers
18.3.2 Tissue- or Lineage-specific Markers
18.3.3 Transcriptomic Analysis
18.3.4 Behavioral Assays
18.4 Cell Imaging
18.4.1 Microscopy
P18.1 Using an Inverted Microscope
18.4.2 Photomicrography
P18.2 Digital Photography on a Microscope
18.4.3 Live-cell Imaging
18.4.4 High-resolution Imaging
18.5 Cell Staining
18.5.1 Preparation of Cultures for Staining
18.5.2 Histological Stains
P18.3 Staining with Giemsa
P18.4 Staining with Crystal Violet
18.5.3 Immunocytochemistry
S18.1 Time-lapse Video Recording
S18.2 Preparation of Suspension Cultures for Cytology by Cytocentrifuge
S18.3 Immunofluorescence Using Chambered Slides
19 Quantitation and Growth Kinetics
19.1 Cell Counting
19.1.1 Manual Cell Counting
P19.1 Cell Counting by Hemocytometer
19.1.2 Automated Cell Counting
19.1.3 Counting Adherent Cells
19.1.4 Cell Weight and Packed Cell Volume (PCV)
19.2 Cell Viability
19.2.1 Dye Exclusion Assays
P19.2 Cell Counting Using Trypan Blue
19.2.2 Dye Uptake Assays
19.3 Cell Proliferation
19.3.1 The Growth Curve
19.3.2 Experimental Design
P19.3 Generating a Growth Curve Using Multiwell Plates
19.3.3 Parameters Derived from the Growth Curve
19.4 Cloning Efficiency
19.4.1 Clonogenic Assays
P19.4 Clonogenic Assay for Attached Cells
19.4.2 Colony Counting
19.4.3 Analysis of Colony Formation
19.5 DNA Synthesis
19.6 Cell Cycle Analysis
S19.1 Estimation of Viability by Dye Uptake
S19.2 Generating a Growth Curve Using Flasks
S19.3 Microautoradiography of Cultured Cells
VI PHYSICAL AND GENETIC MANIPULATION
20 Cell Cloning and Selection
20.1 Terminology: Cloning and Selection
20.2 Cloning by Limiting Dilution
20.2.1 Dilution Cloning in Dishes
P20.1 Dilution Cloning
20.2.2 Dilution Cloning in Microwell Plates
20.3 Cloning in Suspension
20.3.1 Soft Agar
P20.2 Cloning in Agar
20.3.2 Methylcellulose (Methocel)
P20.3 Cloning in Methocel
20.4 Selection of Clones
20.4.1 Adherent Clones
P20.4 Isolation of Adherent Clones with Cloning Rings
20.4.2 Suspension Clones
P20.5 Isolation of Suspension Clones
20.5 Replica Plating
20.6 Stimulation of Cloning Efficiency
20.6.1 Cloning using Conditioned Medium
P20.6 Preparation of Conditioned Medium
20.6.2 Cloning on Feeder Layers
P20.7 Preparation of Feeder Layers
20.6.3 Optimization of Clonal Growth
20.7 Selective Culture Conditions
20.7.1 Selective Media and Inhibitors
20.7.2 Selective Substrates
20.7.3 Selection by Adhesion and Detachment
20.7.4 Selection by Anchorage-independent Growth
20.8 Conclusions: Cloning and Selection
21 Cell Separation and Sorting
21.1 Cell Density and Isopycnic Centrifugation
21.1.1 Density Gradient Centrifugation
P21.1 Cell Separation by Centrifugation on a Density Gradient
21.1.2 Optimization of Density Gradients
21.2 Cell Size and Sedimentation Velocity
21.2.1 Velocity Sedimentation at Unit Gravity
21.2.2 Centrifugal Elutriation
21.3 Magnetic Separation and Sorting
P21.2 Magnet-activated Cell Sorting (MACS)
21.4 Fluorescence-activated Cell Sorting (FACS)
21.5 Microfluidic Sorting
M21.1 Microfluidic Cell Culture
21.6 Conclusions: Sorting and Separation
22 Genetic Modification and Immortalization
22.1 Gene Delivery
22.1.1 Transfection with Calcium Phosphate
P22.1 Calcium Phosphate Coprecipitation
22.1.2 Transfection with Cationic Lipids and Polymers
P22.2 Optimization of Lipofection
22.1.3 Electroporation
22.1.4 Viral Transduction
22.2 Gene Editing
22.2.1 Zinc Finger Nucleases (ZFNs)
22.2.2 Transcription Activator-like Effector Nucleases (TALENs)
22.2.3 CRISPR/Cas RNA-guided Nucleases
P22.3 Delivery of CRISPR/Cas9 RNP using Electroporation
22.3 Immortalization
22.3.1 Early Immortalization Strategies
22.3.2 Immortalization using Viral Genes and Oncogenes
22.3.3 Immortalization using Telomerase
P22.4 Immortalization using hTERT Transfection
22.3.4 Conditional Reprogramming
22.4 Screening and Artifacts
22.4.1 Selection of Modified Cells
22.4.2 Toxicity
22.4.3 Indels and Rearrangements at the Target Site
22.4.4 Off-target Effects
22.4.5 Oncogenesis
S22.1 Fibroblast Immortalization using SV40 TAg
VII STEM CELLS AND DIFFERENTIATED CELLS
23 Culture of Stem Cells
23.1 Terminology: Stem Cells
23.2 Embryonic Stem Cells (ESCs)
23.2.1 Mouse (mESCs)
23.2.2 Human (hESCs)
23.2.3 Other Species
23.3 Induction of Pluripotency
P23.1 Generation of iPSCs using Sendai Virus Vectors
23.4 Human Pluripotent Stem Cell (hPSC) Lines
23.4.1 Evolution in Culture of hPSCs
23.4.2 Culture Conditions
23.4.3 Feeding and Subculture
P23.2 Subculture in Chemically Defined Conditions
23.4.4 Cryopreservation and Thawing
P23.3 Cryopreservation using ROCK Inhibitor
23.5 Perinatal Stem Cells
23.6 Adult Stem Cells
23.7 Stem Cell Characterization and Banking
23.8 Conclusions: Culture of Stem Cells
S23.1 Derivation and Primary Culture of Mouse Embryonic Stem Cells (mESCs)
S23.2 Propagation of Mouse Embryonic Stem Cell (mESC) Lines
S23.3 Derivation and Culture of Human Embryonic Stem Cells (hESCs)
S23.4 Culture of Amniocytes
S23.5 Mesenchymal Stromal Cell (MSC) Production from Human Bone Marrow
24 Culture of Specific Cell Types
24.1 Specialized Cells and their Availability
24.2 Epithelial Cells
24.2.1 Epidermis
24.2.2 Cornea
24.2.3 Oral Epithelium
24.2.4 Bronchial and Tracheal Epithelium
24.2.5 Gastrointestinal Tract
24.2.6 Liver
24.2.7 Pancreas
24.2.8 Breast
24.2.9 Cervix
24.2.10 Prostate
24.3 Mesenchymal Cells
24.3.1 Connective Tissue
24.3.2 Adipose Tissue
24.3.3 Muscle
24.3.4 Cartilage
24.3.5 Bone
24.3.6 Endothelium
24.4 Neuroectodermal Cells
24.4.1 Neurons
24.4.2 Glial Cells
24.4.3 Endocrine Cells
24.4.4 Melanocytes
24.5 Hematopoietic Cells
24.5.1 Lymphoid Cells
24.5.2 Macrophages and Myeloid Cells
24.5.3 Erythroid Cells
24.5.4 Hybridoma Cells
24.5.5 Chimeric Antigen Receptor (CAR) T-cells (CAR T-cells)
24.6 Culture of Cells from Poikilotherms
24.6.1 Fish Cells
24.6.2 Insect Cells
S24.1 Culture of Epidermal Keratinocytes
S24.2 Culture of Corneal Epithelial Cells
S24.3 Culture of Oral Keratinocytes
S24.4 Culture of Human Bronchial Epithelial Cells
S24.5 Isolation and Culture of Colonic Crypts
S24.6 Isolation of Rat Hepatocytes
S24.7 Culture of Pancreatic Epithelium
S24.8 Preparation of Mammary Epithelial cells from Reduction Mammoplasty Specimens
S24.9 Culture of Cervical Epithelium
S24.10 Culture of Rat Prostatic Epithelial Cells
S24.11 Primary Culture of Adipose Cells
S24.12 Culture of Myoblasts from Adult Skeletal Muscle
S24.13 Single Myofiber Culture from Skeletal Muscle
S24.14 Culture of Chondrocytes in Alginate Beads
S24.15 Isolation and Culture of Vascular Endothelial Cells
S24.16 Culture of Rat Cerebellar Granule Cells
S24.17 Culture of Human Astrocytes
S24.18 Culture of Rat Olfactory Ensheathing Cells (OECs)
S24.19 Culture of Melanocytes
S24.20 Preparation and Stimulation of Lymphocytes
S24.21 Production of Monoclonal Antibodies by the B-cell Targeting (BCT) Technique
S24.22 Production of Monoclonal Antibodies by the Stereospecific Targeting (SST) Technique
25 Culture of Tumor Cells
25.1 Challenges of Tumor Cell Culture
25.2 Primary Culture of Tumor Cells
25.2.1 Selection of Representative Cells
P25.1 Freezing Tumor Biopsies
25.2.2 Disaggregation of Tumor Samples
25.3 Development of Tumor Cell Lines
25.3.1 Subculture of Primary Tumor Cultures
25.3.2 Continuous Tumor Cell Lines
25.3.3 Validation of Tumor Cell Lines
25.3.4 Characterization of Tumor Cell Lines
25.4 Selective Culture of Tumor Cells
25.4.1 Selective Media and Techniques
25.4.2 Confluent Feeder Layers
P25.2 Growth on Confluent Feeder Layers
25.4.3 Suspension Cloning
25.4.4 Spheroid Culture
25.4.5 Xenografts
25.5 Specific Tumor Types
25.5.1 Carcinoma
25.5.2 Sarcoma
25.5.3 Melanoma
25.5.4 Lymphoma and Leukemia
25.5.5 Glioma
25.6 Cancer Stem Cells (CSCs)
M25.1 Culture of Cancer Stem Cells
S25.1 Culture of Colorectal Tumors
S25.2 Culture of Mammary Tumor Cells
S25.3 Establishment of Continuous Cell Lines from Leukemia-Lymphoma
26 Differentiation
26.1 In Vitro Models of Differentiation
26.2 Differentiation Status in Culture
26.2.1 Differentiation and Malignancy
26.2.2 Proliferation and Differentiation
26.2.3 Dedifferentiation
26.2.4 Transdifferentiation
26.2.5 Epithelial-mesenchymal Transition (EMT)
26.3 Induction of Differentiation
26.3.1 Exogenous Soluble Factors
26.3.2 Genetic Modifications
26.3.3 Geometry and Polarity
26.3.4 Cell-cell Interactions
26.3.5 Cell-extracellular Matrix (ECM) Interactions
26.3.6 Air-liquid Interface
26.3.7 Biomechanical Regulation
26.4 Practical Aspects
26.5 Ongoing Challenges
26.5.1 Markers of Differentiation
26.5.2 Stem Cell Differentiation
S26.1 Purification of HepaRG Human Hepatocytes
VIII MODEL ENVIRONMENTS AND APPLICATIONS
27 Three-dimensional Culture
27.1 Terminology: 3D Culture
27.2 Technologies for 3D Culture
M27.1 Advances in Technologies Enabling Three-dimensional Cell Culture and the Formation of Tissue-like Architecture In Vitro
27.3 Benefits and Limitations of 3D Culture
27.4 Scaffold-free 3D Culture Systems
27.4.1 Spheroid Culture
P27.1 Tumor Spheroid Formation and Embedding
27.4.2 Dynamic Culture Systems
27.5 Scaffold-based 3D Culture Systems
27.5.1 Overlay, Embedding, and Encapsulation
27.5.2 Filter Well Inserts
P27.2 Culture Using Filter Well Inserts
27.5.3 Hollow Fiber Systems
27.5.4 Microcarriers and Macrocarriers
27.6 Organoid Culture
27.7 Organotypic Culture
27.7.1 Tissue Equivalents
27.7.2 Tissue Engineering
27.8 Organ Culture
27.9 Characterization of 3D Cultures
S27.1 3D Spheroid Culture Using an Agar Underlay
S27.2 In Vitro Angiogenesis Assay
S27.3 Organ Culture from Chick Embryo
28 Scale-up and Automation
28.1 Terminology: Scale-up and Bioreactors
28.2 Scale-up in Suspension
28.2.1 Spinner Culture
28.2.2 Single-use Bioreactor Systems
28.2.3 Scaffold-free Perfusion Bioreactors
28.2.4 Other Bioreactor Systems for Suspension Culture
28.3 Scale-up in Monolayer
28.3.1 Roller Culture
28.3.2 Multisurface Propagators
P28.1 Handling a Nunc Cell Factory
28.3.3 Microcarrier Culture
28.3.4 Scaffold-based Perfusion Bioreactors
28.4 Monitoring and Process Control
28.5 Scale-up for Manufacture
M28.1 Culture Scale-up and Bioreactors
28.6 High-throughput Screening
28.7 Automation and Bioprinting
28.7.1 Automation of Culture Handling
28.7.2 Automation of Cell-based Assays
28.7.3 Three-dimensional (3D) Bioprinting
S28.1 Roller Bottle Culture
29 Toxicity Testing
29.1 In Vitro Toxicity Testing
29.1.1 Applications
29.1.2 Limitations
29.1.3 Requirements
29.2 Cytotoxicity Assays
29.2.1 Selecting a Cytotoxicity Assay
29.2.2 Assays Based on Cell Metabolism
P29.1 MTT-based Cytotoxicity Assay
29.2.3 Assays Based on Cell Death
29.2.4 Assays Based on Cell Survival
29.2.5 Analysis of Cytotoxicity Assays
29.3 Genotoxicity Assays
29.4 Carcinogenicity Assays
29.5 Advanced Models for Toxicity Testing
29.5.1 3D Models for Eye and Skin Irritation
29.5.2 Organ-on-chip Technologies
S29.1 Clonogenic Assay for Cytotoxicity Testing
IX TEACHING AND TROUBLESHOOTING
30 Training
30.1 Training Principles
30.1.1 Roles and Responsibilities
30.1.2 Induction
30.1.3 Training Documents
30.1.4 Hands-on Training
30.2 Training Programs
30.2.1 Topics
30.2.2 Exercises
S30.1 Washing and Sterilizing Glassware
S30.2 Preparation and Sterilization of Water
S30.3 Preparation and Sterilization of Dulbecco's Phosphate-buffered Saline without Ca2+ and Mg2+ (DPBS-A)
S30.4 Preparation of pH Standards
S30.5 Preparation of Basal Medium from Powder and Sterilization by Filtration
S30.6 Pipetting and Transfer of Fluids in a Biological Safety Cabinet (BSC)
S30.7 Preparation of Complete Culture Medium
S30.8 Observation of Cultured Cells
S30.9 Feeding Adherent Cultures
S30.10 Counting Cells by Hemocytometer and Automated Cell Counter
S30.11 Subculture of Adherent Cultures
S30.12 Subculture of Suspension Cultures
S30.13 Cryopreservation of Cultured Cells
S30.14 Thawing of Frozen Cryovials
S30.15 Primary Culture
31 Problem Solving
31.1 Microbial Contamination
31.1.1 Type of Microbial Contamination
31.1.2 Contamination is Limited to One Person
31.1.3 Contamination is Widespread
31.1.4 Problems with Laminar Flow or Air Quality
31.2 Cross-contamination and Misidentification
31.3 Chemical Contamination
31.4 Slow Cell Growth
31.4.1 Problem is Limited to One Person
31.4.2 Problem is Widespread
31.5 Abnormal Cell Appearance
31.6 Problems with Materials
31.6.1 Culture Vessels
31.6.2 Medium Formulation and Preparation
31.6.3 Medium Stability and Storage
31.6.4 Water Quality
31.7 Problems with Primary Culture
31.7.1 Suspected Contamination
31.7.2 Poor Take in Primary Culture
31.7.3 Incorrect Phenotype after Primary Culture
31.8 Problems with Feeding or Subculture
31.8.1 pH after Feeding
31.8.2 Poor Take after Subculture
31.8.3 Uneven Growth after Subculture
31.9 Problems with Cryopreservation
31.9.1 Loss of Frozen Stocks
31.9.2 Poor Viability after Thawing
31.9.3 Changed Appearance after Thawing
31.10 Problems with Cloning
31.10.1 Too Few Colonies per Dish
31.10.2 Too Many Colonies per Dish
31.10.3 Nonrandom Distribution of Colonies
31.10.4 Incubation of Cloning Dishes
32 In Conclusion
Appendix I Glossary
Appendix II Calculations and Preparation of Reagents
Appendix III Media Formulations
Index
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