Animal Cell and Tissue Culture

ABOUT THE BOOK: Man is considered to be the most powerful creature in universe, but he depends totally on the biological entities for his all nutritional needs. Biotechnology use biological entities for the betterment of human kind. Biotechnology derived from fusion of biology and technology, is a very vast subject which includes Plant cell and Tissue culture, Animal cell and tissue culture, Transgenic, Molecular biology, Recombinant DNA technology, Genetic engineering, Medical biotechnology, Fermentation technology, Microbiology, Enzyme technology, Biofuels, Environmental biotechnology, Bioinformaticsand Nanotechnology.
It may be pointed out that use of biological entities is not new. Ever since establishment of civilization man is using biological organisms to satisfy his needs. But this cannot be regarded as biotechnology since production technology pertaining to agriculture and animal husbandry etc. has now become distinct disciplines. However exploitation of plant and animal cell in vitro as well as their constituents to generate some useful product constitute biotechnology.
Animal cell and tissue culture is an integral part of Biotechnology and this book covers all the aspects of animal cell culture. Animal cells are used for making new vaccines, specific animal proteins such as intergerons, blood factors "and hormones, monoclonal antibodies for use as diagnostic and therapeutics, gene probes as diagnostic too, enzymes and last but not the least many new and important compounds.
This book is beneficial to all students of under graduation as well as post graduation of all universities.
This book comprises 13 chapters covering all aspects of Animal Cell Biotechnology and very recent area of research i.e. Tissue engineering has also been included with updated text materials and graphics.
Chapter 1 of this book describes the general introduction and basics about animal cell and tissue culture. Chapter 2, 3 and 4 describes how to get start with animal cell culture and to establish cell lines for further studies use? Chapter 5 and 6 tells about the selection and preservation of animal cells which becomes necessary if we have to deal with genetically modified / clones cells. Chapter 7 gives an idea about the feasibility of scale up process and chapter 8 covers all aspects of fermentors used in scale up process of animal cells. Chapter 9 explains the most extensive use of animal cell culture i.e. hybridoma technology and production of monoclonal antibodies. Genetic manipulation of animal cells (transgenesis), a relatively new field is dealt in chapter 10 and its applications are given in chapter 11 which are very useful to all students of biotechnology as well as other students of relevant subjects.
Chapter 12 is the most important, which enhances the importance of this book. Tissue engineering is an emerging area of research in medical biotechnology and has shown good future for transplantation biology. Similarly chapter 13 is comprising new field of biotechnology; the Gene therapy. Gene therapy has already proven its importance in the treatment of various diseases like SCID, cystic fibrosis etc.

AUTHOR: Shviangi Mathur | ISBN: 9788177542974 | YEAR: 2009 | PAGES: 266 | SIZE: 19 X 25 X 2 cm. | BINDING: Hard | LANGUAGE: English

ABOUT THE AUTHOR: Ms. Shivangi Mathur did M. Sc. Biotechnology from Rajasthan University, Jaipur and P. G. Diploma in Bioinformatics from Bioinformatics Institute, Noida. She has been trained at National Research Centre on Camel (NRCC), Bikaner on various aspects of Biotechnology like RFLP, RAPD, PCR and Microsatellite DNA analysis. She has presented papers in various International and National seminars and symposia. After working in various educational institutions in Jaipur, currently she is teaching Biotechnology at S. S. Jain Subodh Girls College, Jaipur. She is an author of the book Biotechnology: Fundamentals and Applications (in Hindi) in collaboration with Dr. S. S. Purohit.

CONTENTS:
1. CELLS IN CULTURE: AN INTRODUCTION
    a) History
    b) Advantages and Disadvantages of Tissue Culture Methods
        i. Advantages
        ii. Disadvantages
    c) Biology of Cells in Culture
        i. Origin and Characterization
        ii. Differentiation
        iii. Kinetics of Cell Growth
        iv. Genetics of Cultured Cells
        v. Laboratory Requirements for Animal Cell Culture
    d) Choice of Materials
        i. Cell Type
        ii. Tissue Source
        iii. Subculturing
        iv. Selection of Medium
        v. Gas Phase
        vi. Selection of Substrate
        vii. Size of Culture Vessel
        viii. Feeder Layer on Substrate
    e) Applications of Animal Cell Culture
        i. Monoclonal Antibodies
        ii. Recombinant Proteins
        iii. Gene Targeting
        iv. Cytotoxicity Testing
        v. Tissue Grafting
        vi. Amniocentesis, Infertility and Embryo Transplantation
    f) Problems Associated with Tissue Culture
        i. Contamination
        ii. Mycoplasma
        iii. Cross Contamination
    g) Instability
    h) Ethical Considerations
2. TISSUE CULTURE MEDIUM
    a) Introduction
        i. Natural Media
        ii. Biological Fluids
        iii. Tissue Extracts
        iv. Chemically Defined Medium
        v. Considerations for Designing of Media
        vi. Surface Tension and Foaming
        vii. CO, and Bicarbonates
        viii. Temperature
    b) Media Designing
    c) Characteristics / Composition of Media
        i. Energy Source
        ii. Amino Acids
        iii. Vitamins
        iv. Minerals
        v. Antibiotics
        vi. Serum
3. INITIATION OF CELL CULTURE PRIMARY CULTURE
    a) Introduction
    b) Preparation and Sterilization of Glassware and Apparatus
    c) Preparation and Sterilization of Reagents and Media
    d) Preparation of Animal Material
        i. Primary Culture
        ii. Mechanical Disaggregation
        iii. Enzymatic Disaggregation
        iv. Disaggregation by Trypsin (for Embryonic Tissue) or Trypsinization
        v. Warm Trypsinization
        vi. Cold Trypsinization
        vii. Disaggregation by Collagenase (for Embryonic, Normal and Malignant Tissues)
    e) Culture
        i. Primary Explantation Techniques
        ii. Slide or Coverslip Cultures
        iii. Flask Cultures
        iv. Test Tube Cultures
    f) Organ Culture
        i. Characteristics of Organ Culture
        ii. Limitation of Organ Culture
        iii. Culture of Embryonic Organs
        iv. Organ Cultures on Plasma Clots
        v. Organ Cultures on Agar
    g) Whole Embryo Culture
        i. Culture of Chick Embryo (Spratt, 1956)
    h) Histotypic Cultures
        i. Spheroides
        ii. Gel and Sponge Technique
        iii. Hollow Fibers
    i) Applications of Cell Cultures
        i. Animal Tissue Cultures in Biomdical Research: Genetics
        ii. Tissue Cultures in Biomedical Research: Virology and Host-Parasite
            1. Relationships
        iii. Tissue Cultures in Biomedical Research: Cancer
4. CELL LINES
    a) Need of Subculturing
    b) Subculturing
        i. Immortalization of Cell Lines
        ii. Designation of Cell Line
    c) Selection of Cell Lines
        i. Finite Cell Line I Continuous Cell Line
        ii. Species
        iii. Availability
        iv. Normal or Transformed Line
        v. Growth Characteristics:
        vi. Stability
        vii. Validation
        viii. Control of Cell Line
    d) Routine Maintenance of Cell Lines
        i. Cell Morphology
        ii. Replacement of Medium
    e) Protocol
        i. Feeding the Culture
        ii. Surface Area, Volume and Depth
        iii. Subculture
        iv. Criteria for Subculture
        v. Procedure for Subculture
        vi. Subculture of Suspension Culture
    f) Protocol 2
        i. Subculture of Suspension
    g) Comparison of Monolayer and Suspension Culture
5. SELECTION AND CLONING OF CELLS
    a) Introduction
        i. Objectives of Cloning
        ii. Cloning of Monolayer and Suspension Culture
        iii. Dilution Cloning
        iv. Factors affecting Clonal Growth
        v. Cloning in Suspensions
        vi. Cloning in Methocel
        vii. Selection and Isolation of Clones
        viii. Other Isolation Techniques
        ix. Factors affecting Cell Adhesion
6. CHARACTERIZATION AND PRESERVATION OF ANIMAL CELLS
    a) Need of Characterization
        i. Species Verification
        ii. Intraspecies Contamination
        iii. Microbial Contamination
        iv. To Check Status of Cells83
        v. To Check Genetic Stability of Cells
    b) Techniques of Characterization
        i. Techniques for Species Verification
    c) Chromosome Banding
        i. Protocol: Chromosome Banding
        ii. DNA Content
        iii. DNA Hybridization
        iv. Isoenzymology
        v. Protocol 2: Isoenzyme Analysis
        vi. Immunological Tests
        vii. Techniques to Detect Intra Species Contamination
        viii. Techniques for Characterizing Cell Types
        ix. Techniques to Detect Microbial Contamination
        x. Preservation of Animal Cell Lines
        xi. Steps of Cryopreservation
        xii. Evaluation of Cell Viability
    d) Thawing
        i. Cell Banks
7. SCALE UP OF ANIMAL CELL CULTURE
    a) Practical Considerations during Animal Cell Culture
        i. Culture Parameters
        ii. Medium
        iii. Non Nutritional Medium Supplements
        iv. Oxygen
        v. Redox Potential
        vi. Growth Kinetics
        vii. Culture Vessel and Growth Surfaces
        viii. Cell Quantification
    b) Scale up Process
    c) Scale up of Monolayer
        i. Nunc Cell Factory
        ii. Multitray Disks, Spirals and Tubes
        iii. Roller Bottles I Tubes
        iv. Microcarriers
        v. Perfused Monolayer Culture
        vi. Microencapsulation
    d) Scale up of Suspension
        i. Protocol: Suspension Cultures
        ii. Continuous Culture
    e) Mixing and Aeration in Suspension
        i. Airlift Fermenters
        ii. Perfused Suspension Culture
        iii. Rotating Chambers
        iv. Fluidized Bed Reactors for Suspension Culture
    f) On Line Monitoring of Cell Culture
8. FERMENTORS
    a) Continuous Culture
    b) The Components of a Fermentor
    c) Parts of Fermentors
        i. Vessel
    d) Peripheral Parts and Accessories
        i. Reagent Pumps
        ii. Medium Feed Pumps and Reservoir Bottles
        iii. Rotameter I Gas Supply
        iv. Sampling Device
    e) Alternative Vessel Designs
        i. Air Lift
        ii. Fluidized Bed
        iii. Hollow Fibre
        iv. In Situ Sterilizable Fermentors
        v. Containment
    f) Additional Accessories and Peripherals
        i. Feed Pumps
    g) Exit Gas Analysis
        i. lnfra-Red Carbon Dioxide Analyzer
        ii. Paramagnetic Oxygen Analyzer
        iii. Mass Spectrometer
    h) Types of Reactors
        i. Mechanically Agitated Stirred Tank Reactors
        ii. Air-lift Bioreactor and Modified Air Lift Bioreactors
        iii. Novel Seesaw Bioreactor
    i) Different Types of Fermentor Instrumentation
        i. Analogue Controllers - Rack System
        ii. Analogue Controllers - Separate Modules in Housings
        iii. Digital Controllers - Embedded Microprocessor
        iv. Digital Controllers - Process Controllers
        v. Digital Controllers - Direct Computer Control
    j) Common Measurement and Control Systems
        i. Speed Control
        ii. Temperature Control
        iii. Control of Gas Supply
        iv. Control of Dissolved Oxygen
        v. Antifoam Control
    k) Transfer of Oxygen
    l) Maintenance of Aseptic Conditions
9. HYBRIDOMA TECHNOLOGY AND MONOCLONAL ANTIBODIES
    a) Hybridoma
        i. Limitation of Traditional Method of Antibody Production
        ii. Advantages and Disadvantages of MABso over Polyclonal Antibodies
        iii. Types of MAbs
        iv. Chimeric MAbs
    b) Hybridoma Technology
        i. The Basis of Hybridoma Technology
    c) Production of Monoclonal Antibodies
        i. Choice of Fusion Partners
        ii. Immunization of Mice
        iii. Making Hybridomas
        iv. Screening Procedures
        v. Long Term Storage
    d) Hybridomas from Different Species
    e) Production of MAbs through Genetic Engineering
    f) Alternative to Hybridoma
    g) Production of Human/Humanized Antibodies
    h) Application of MAbs
        i. Diagnosis Screening and Therapy
    i) Monoclonal Antibodies as Enzymes (Abzymes)
    j) Vaccine Production
    k) Purification and Quantitation of Compounds
        i. Raising MAbs
        ii. Purification and Quantitation
        iii. Radio Immunoassay (RIA)
    l) Cytogenetic Analysis
10. GENETIC MANIPULATION OF CELLS: TRANSGENESIS
    a) Why Animal Cells are Chosen for Genetic Manipulation
    b) Basics of Genetic Manipulation
        i. Isolation of Gene
        ii. Introduction of Gene into Suitable Vector
        iii. Introduction of Recombinant Vector into Cells
        iv. Calcium Phosphate Transfection
        v. Lipofection
        vi. Electroporation
        vii. Microinjection
        viii. Retroviral Infection
        ix. Embryonic Stem Cell Transfer
        x. Microprojectile I Biolistic
    c) Screening for Recombinant Cells
        i. Analysis of Transgene Integration
        ii. Detection ofmRNA Expression
        iii. Assay for Protein Expression
    d) Examples of Transgenic Animals
        i. Integration of Microinjected Sequences into the Embryo: A Case Study of Mice
        ii. Genotyping Transgenic Mice by PCR
    e) Specific Applications of Transgenic Mice
        i. Gene Expression
        ii. Transgenic Mice as Models for Genetic Engineering
    f) Transgenic Swine
    g) Production of Sheep Transgenic for Growth Hormone Genes
        i. Insertion of Genes
        ii. Expression of Transgenes
    h) Production of Transgenic Cattle by Pronuclear Injection
        i. Collection of Embryos
        ii. Pronuclear Injection
        iii. Preparation of DNA
        iv. Culture and Transfer of Embryos
        v. Collection and Analysis of Tissue
    i) Methods for the Introduction of Recombinant DNA into Chicken Embryos
    j) Problems after Developing Transgenic Animals
        i. The Nature of Patents
11. APPLICATIONS OF TRANSGENIC ANIMALS
    a) Transgenic Animal Technology
    b) Genuine Transgenic Animals and their Applications
        i. Enhanced and Tissue-Specific Transgene Expression
        ii. Production of Sheep Transgenic for Growth Hormone Genes
        iii. Production of Transgenic Cattle by Pronuclear Injection
        iv. Methods for the Introduction of Recombinant DNA into Chicken Embryos
    c) Present States of Art
    d) Gene Transfer by Microinjection
    e) Other Methods
    f) Use for Medical and Therapeutic Purposes
        i. Recombinant Protein Production
        ii. Xenotransplantation
    g) Genetic Engineering- Single Genes
    h) Gene Mapping
    i) Artificial Insemination and Germ Cell Storage
    j) Ectogensis
    k) Amniocentesis
        i. Significance of Amniocentesis
    l) Transplantation
    m) Cloning Technique
    n) Gene Therapy
    o) Genetic Counselling
    p) In Vitro Fertilization and Embryo Transfer
        i. Surrogate Motherhood
        ii. In Vitro Fertilization and Embryo Transfer for Improved Breeding
            1. Programmes
        iii. Success of Embryo Transplantation
        iv. Objectives and Applications of ET
        v. Superovulation
        vi. Physiological Basis of Superovulation
        vii. Factors Which Influence the Superovulatory Response
        viii. Superovulation using Pmsg
        ix. Freezing of Embryos
        x. Frozen Embryos
        xi. Embryo Sexing
        xii. Success in Pre-Determining the Sex
        xiii. Micromanipulation of Embryos
    q) Advantages of Cell Manipulation Technique
        i. Simple Method of Splitting Embryos
    r) Techniques of Nuclear Transplantation
        i. Sources of Eggs and Embryos
    s) Gene Injection (Introduction of Foreign Cloned Genes)
        i. Dense Cytoplasm and Gene Injection
    t) Selective Animal Breeding
        i. Intensity of Selection and Reproductive Rate
    u) Potential
    v) Hazards of Artificial Breeding
        i. Animals as Tennis Balls?
        ii. Down on the Farm
        iii. Time to Decide
12. TISSUE GRAFTING AND ENGINEERING
    a) Introduction
    b) Historical Perspective
        i. Principle
        ii. Prerequisite for Tissue Engineering
        iii. Semi-Natural and Natural Substrate
        iv. Synthesis of Tissue Engineering Scaffolds
        v. Cell Sources
        vi. Assembly Methods
    c) Some Examples of Tissue Engineering
        i. Use of Biological Film as a Cultured Epidermal Autograft
        ii. Tissue Engineering Peripheral Nerve Implants
        iii. Tissue Engineering for Vascular Graft
        iv. Advantages and Disadvantages of Tissue Engineering
        v. Ethical Issues
        vi. Future Prospects
13. GENE THERAPY
        i. How does Gene Therapy Work?
        ii. Gene Therapy in the Treatment of Cancer
        iii. Main Approaches of Gene Therapy
        iv. Gene Therapy and Transfer of Genes Into Cells
    a). Viruses used as Gene Therapy Vectors
        v. Retroviruses
        vi. Adenoviruses
        vii. Adeno-Associated Viruses
        viii. Herpes Simplex Viruses
    b) Current Status of Gene Therapy Research
    c) Gene Therapy Factors and Genetic Disease
        i. Short Lived Nature of Gene Therapy
        ii. Immune Response
        iii. Problems with Viral Vectors
        iv. Multigene Disorders
    d) Recent Developments in Gene Therapy
        i. Subtle Gene Therapy Tackles Blood Disorder
    e) DNA Nanoballs Boost Gene Therapy
    f) Risks Associated with Gene Therapy Trials
    g) Precautions for Gene Therapy
REFERENCES
SUBJECT INDEX