FRESHNEY’S CULTURE OF ANIMAL CELLS

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:

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 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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