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| List of protocols | |
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| Abbreviations | |
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| The cell culture laboratory | |
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| Animal cell culture: past, present, and future | |
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| The laboratory | |
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| Design concepts and layout | |
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| Services | |
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| Equipping the laboratory | |
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| Major items of equipment and instrumentation | |
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| Culture plasticware and associated small consumable items | |
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| Washing reusable tissue culture equipment | |
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| Soaking | |
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| Washing | |
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| Pipette washing | |
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| General care and maintenance of the tissue culture laboratory | |
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| Acknowledgements | |
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| References | |
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| Sterilization | |
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| Introduction | |
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| What is sterilization? | |
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| The importance of sterility in cell culture | |
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| The use of antibiotics | |
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| Basic principles | |
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| Wet heat at up to 100[degree]C | |
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| Theory | |
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| Pasteurization | |
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| 100[degree]C | |
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| Wet heat above 100[degree]C and autoclaving | |
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| Theory | |
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| Steam | |
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| Types of autoclave | |
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| Preparation of the load and operation of the autoclave | |
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| Autoclave testing | |
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| Dry heat | |
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| Incineration | |
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| Hot-air ovens | |
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| Irradiation | |
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| Ultraviolet light | |
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| Gamma rays | |
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| Chemical sterilization | |
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| Fumigation | |
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| Liquid disinfectants | |
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| Filtration | |
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| Filters for bacteria and fungi | |
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| Filters for viruses | |
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| HEPA filters | |
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| Viruses and prions | |
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| Virus elimination | |
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| Prions | |
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| Safety in the laboratory | |
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| Good Manufacturing Practice and Good Laboratory Practice | |
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| Acknowledgements | |
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| References | |
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| Culture media | |
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| Introduction | |
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| Basal media | |
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| Types of basal media | |
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| Constituents of basal media | |
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| Choice of basal medium | |
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| Preparation of basal medium | |
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| Serum | |
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| Why use serum? | |
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| Types of serum | |
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| Constituents of serum | |
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| Potential problems with the use of serum | |
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| Sourcing and selection of serum | |
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| Serum storage and use | |
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| Replacement of serum in media | |
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| Reduced-serum media for early-passage normal human cells | |
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| Serum-free media (SFM) | |
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| Design of SFM | |
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| Influence of cell culture systems on the choice of medium | |
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| Batch or perfusion cultures | |
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| Anchorage-dependent cells | |
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| Stirred suspension cultures | |
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| High density culture systems | |
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| Very low density cultures | |
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| In-house medium development and production versus commercial supply | |
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| Economic considerations | |
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| Development of a dedicated medium | |
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| Quality assurance and control | |
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| References | |
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| Microscopy of living cells | |
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| Introduction | |
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| The modern light microscope | |
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| Basic scheme | |
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| Properties of light | |
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| Magnification | |
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| Resolution | |
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| Aberrations and their corrections | |
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| Illumination | |
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| Maintenance | |
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| Microscopic techniques | |
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| Bright field | |
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| Oblique illumination | |
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| Dark field | |
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| Phase-contrast | |
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| Differential interference contrast (DIC) | |
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| Hoffman modulation contrast | |
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| Reflection contrast | |
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| Varel contrast | |
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| Polarizing microscopy | |
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| Interference microscopy | |
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| Fluorescence microscopy | |
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| Specimen preparation | |
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| Culture flasks and Petri dishes | |
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| Coverslips | |
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| Chambers | |
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| Interaction with microscopic techniques | |
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| Environmental control for microscopic specimens | |
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| Recording of microscopic images | |
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| Time-lapse imaging | |
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| Analysis of microscopic images | |
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| Image enhancement by deconvolution | |
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| Image processing--extraction of morphometric data | |
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| Data analysis--statistical evaluation | |
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| Data interpretation--simulation modelling | |
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| Acknowledgements | |
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| References | |
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| Basic cell culture technique and the maintenance of cell lines | |
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| Introduction | |
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| The terminology of cell and tissue culture | |
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| Basic components of the cell culture environment | |
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| Culture medium | |
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| Physicochemical factors | |
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| Stationary versus dynamic media supply | |
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| Sterile technique and contamination control | |
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| Working within the laminar flow hood | |
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| Pipetting and prevention of aerosol formation | |
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| Additional considerations for good sterile technique | |
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| Prevention of cellular cross-contamination | |
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| Cell culture at the open bench | |
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| General procedures for the cell culture laboratory | |
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| Maintenance of the laboratory | |
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| Daily inspection of ongoing cultures | |
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| Maintenance of stock cultures | |
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| The culture of attached cells | |
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| Routine culture substrates | |
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| Choice of culture vessels | |
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| Artefacts of cell attachment and growth | |
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| Use of attachment factors and bio-substrates | |
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| Alternative culture substrates | |
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| Three-dimensional matrices | |
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| Culture of cells on microcarriers | |
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| Mass culture systems for adherent cells | |
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| The culture of cells in suspension | |
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| Non-adherent substrates for small-scale culture | |
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| Mass culture of cells in fluid suspension | |
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| Micro-encapsulation | |
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| In vitro cell growth behaviour | |
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| Adaptation to culture | |
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| Phases of cell growth | |
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| Determinations of cell growth data | |
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| Calculation of in vitro age | |
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| Multiplication rate and population doubling time | |
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| Counting cells in suspension | |
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| Counting cells adherent to a substrate | |
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| Phases of the cell cycle | |
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| Cell generation time by time-lapse photomicrography | |
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| Growth fraction | |
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| Expressions of culture 'efficiency' | |
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| Cryopreservation and retrieval of cells from frozen storage | |
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| Purpose of cell banking | |
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| Mechanism of cell freezing, and factors that affect viability | |
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| Supplies and equipment for cell freezing | |
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| Additional comments regarding cryopreservation | |
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| Transportation of cells | |
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| Transporting frozen cells | |
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| Transporting growing cells | |
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| Safety in the cell culture laboratory | |
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| Potential risks in routine cell culture | |
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| Awareness of increased risk associated with human cells | |
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| Classification of cell lines as aetiological agents | |
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| Precautions in handling pathogenic organisms and human cells | |
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| References | |
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| Primary culture and the establishment of cell lines | |
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| Introduction | |
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| Establishment of primary cultures from various sources | |
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| Source of material | |
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| Isolation of cells | |
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| Substrate for attachment | |
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| Culture conditions | |
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| Evolution of primary cultures | |
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| Nomenclature | |
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| Subculture | |
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| Growth phases | |
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| Characteristics of limited lifespan cultures | |
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| Lifespan and senescence | |
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| Phenotype | |
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| Karyotype | |
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| Establishment of continuous cell lines | |
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| Spontaneous | |
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| Chemical transformation | |
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| Viral transformation | |
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| Properties of continuous cell lines | |
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| Aneuploidy | |
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| Heterogeneity and instability | |
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| Differentiated status | |
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| Tumorigenicity | |
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| Cell fusion | |
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| Methods | |
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| Properties of hybrids | |
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| Applications | |
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| Genetic engineering techniques | |
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| Introduction of genes | |
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| Oncogene immortalization | |
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| Safety considerations | |
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| Concluding remarks | |
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| Advantages of cell culture over in vivo experimentation | |
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| Limitations | |
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| Acknowledgements | |
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| References | |
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| Specific cell types and their requirements | |
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| Introduction | |
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| General principles | |
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| Growth requirements of cells in vitro | |
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| Epithelial cells | |
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| Mesenchymal cells | |
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| Neuro-ectodermal cells | |
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| Gonadal cells | |
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| Embryonal stem (ES) cells | |
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| Leukocytes | |
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| Insect cells | |
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| Conclusion | |
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| Acknowledgements | |
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| References | |
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| Cloning | |
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| Introduction | |
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| Development of techniques | |
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| Uses of cloning | |
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| Limitations of cloning | |
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| Special requirements of cells growing at very low densities | |
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| Cell cloning procedures | |
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| Choice of technique | |
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| Methods applicable to both attached and suspension cells | |
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| Methods for attached cells | |
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| Methods for suspension cells | |
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| References | |
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| The quality control of cell lines and the prevention, detection, and cure of contamination | |
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| Introduction | |
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| Obtaining the basic material | |
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| Importance of cell culture collections | |
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| Resource centres | |
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| Cell culture databases | |
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| Quarantine and initial handling of cell lines | |
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| Accessioning scheme | |
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| Laboratory design | |
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| Handling of cell lines | |
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| Production of cell banks | |
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| Microbial quality control | |
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| Sources of microbial contamination | |
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| Testing for bacteria, yeasts, and other fungi | |
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| Testing for mycoplasma | |
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| Virus testing | |
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| Bovine spongiform encephalopathy | |
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| Elimination of contamination | |
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| Authentication | |
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| Isoenzyme analysis | |
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| Cytogenetic analysis | |
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| DNA fingerprinting | |
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| Regulatory aspects | |
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| References | |
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| Good Laboratory Practice in the cell culture laboratory | |
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| What happens if you fall under a bus? | |
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| GLP, an overview | |
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| What is GLP and when should it be used? | |
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| Planning the study | |
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| Performance of the study | |
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| Personnel | |
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| Methods | |
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| Equipment | |
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| Materials | |
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| Monitoring | |
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| Facilities and systems | |
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| Study-specific audits | |
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| Final report audit | |
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| Records | |
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| Advice for researchers creating new cell lines | |
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| Acknowledgements | |
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| References | |
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| List of suppliers | |
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| Culture collections and other resource centres | |
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| Online resources for cell biology | |
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| Index | |