Animal Cell and Tissue Culture

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2 Animal Cell and Tissue Culture Shivangi Mathur Department of Biotechnology S.S. Jain Subodh Girls College, Jaipur AGROBIOS (INDIA)

3 Published by: AGROBIOS (INDIA) Agro House, Behind Nasrani Cinema Chopasani Road, Jodhpur Phone: , Fax: E. mail: Web Site: agrobiosindia.com All Rights Reserved, 2012 First Published: 2006 Reprinted: 2008 Reprinted: 2012 All rights reserved. No part of the book or part thereof, including the title of the book, be reprinted in any from or language without the written permission of the author and the publishers. The copyists shall be prosecuted. ISBN No. (10): ISBN No. (13): Price: Rs / US$ Published by: Dr. Updesh Purohit for Agrobios (India), Jodhpur Lasertypeset at: Yahsee Computers, Jodhpur Cover Design by: Reena Printed at: Hinglaj Offset Printers, Jodhpur

4 PREFACE 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, Bioinformatics and 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 can not be regarded as biotechnology since production technology pertaining to agriculture and animal husbandry etc. has now become distinct disciplines. However exploitation of platn 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 undergraduation 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. In chapter 2, chapter 3 and chapter 4 I have described how to get start with animal cell culture and to establish cell lines for further studies use? Chapter 5 and chapter 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 upprocess and chapter 8 covers all aspects of fermentors used in scale up process of animal cells. Chapter 9 explains the most

5 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 relavant subjects. Chapter 12 is the most important chapter 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 also 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. In sincerely thank all those who have given their valuable time and help in completing this book. Author

6 CONTENTS 1. CELLS IN CULTURE: AN INTRODUCTION... 1 History... 1 Advantages and Disadvantages of Tissue Culture Methods... 2 Advantages... 2 Disadvantages... 2 Biology of Cells in Culture... 2 Origin and Characterization... 2 Differentiation... 3 Kinetics of Cell Growth... 3 Genetics of Cultured Cells... 5 Laboratory Requirements for Animal Cell Culture... 5 Choice of Materials... 7 Cell Type... 7 Tissue Source... 7 Subculturing... 8 Selection of Medium... 8 Gas Phase... 9 Selection of Substrate... 9 Size of Culture Vessel... 9 Feeder Layer on Substrate Applications of Animal Cell Culture Monoclonal Antibodies Recombinant Proteins Gene Targeting Cytotoxicity Testing Tissue Grafting Amniocentesis, Infertility and Embryo Transplantation Problems Associated with Tissue Culture Contamination Mycoplasma Cross Contamination Instability Ethical Considerations... 14

7 2. TISSUE CULTURE MEDIUM Introduction Natural Media Biological Fluids Tissue Extracts Chemically Defined Medium Considerations for Designing of Media Surface Tension and Foaming CO 2 and Bicarbonates Temperature Media Designing Characteristics / Composition of Media Energy Source Amino Acids Vitamins Minerals Antibiotics Serum INITIATION OF CELL CULTURE- PRIMARY CULTURE Introduction Preparation and Sterilization of Glassware and Apparatus Preparation and Sterilization of Reagents and Media Preparation of Animal Material Primary Culture Mechanical Disaggregation Enzymatic Disaggregation Disaggregation by Trypsin (for Embryonic Tissue) or Trypsinization Warm Trypsinization Cold Trypsinization Disaggregation by Collagenase (for Embryonic, Normal and Malignant Tissues) Culture Primary Explantation Techniques Slide or Coverslip Cultures Flask Cultures Test Tube Cultures Organ Culture Characteristics of Organ Culture Limitation of Organ Culture Culture of Embryonic Organs Organ Cultures on Plasma Clots Organ Cultures on Agar Whole Embryo Culture Culture of Chick Embryo (Spratt, 1956) Histotypic Cultures... 51

8 Spheroides Gel and Sponge Technique Hollow Fibers Applications of Cell Cultures Animal Tissue Cultures in Biomdical Research: Genetics Tissue Cultures in Biomedical Research: Virology and Host-Parasite Relationships Tissue Cultures in Biomedical Research: Cancer CELL LINES Need of Subculturing Subculturing Immortalization of Cell Lines Designation of Cell Line Selection of Cell Lines Finite Cell Line / Continuous Cell Line Species Availability Normal or Transformed Line Growth Characteristics Stability Validation Control of Cell Line Routine Maintenance of Cell Lines Cell Morphology Replacement of Medium Protocol Feeding the Culture Surface Area, Volume and Depth Subculture Criteria for Subculture Procedure for Subculture Subculture of Suspension Culture Protocol Subculture of Suspension Comparison of Monolayer and Suspension Culture SELECTION AND CLONING OF CELLS Introduction Objectives of Cloning Cloning of Monolayer and Suspension Culture Dilution Cloning Factors Affecting Clonal Growth Cloning in Suspensions Cloning in Methocel Selection and Isolation of Clones... 77

9 Other Isolation Techniques Factors Affecting Cell Adhesion CHARACTERIZATION AND PRESERVATION OF ANIMAL CELLS Need of Characterization Species Verification Intraspecies Contamination Microbial Contamination To Check Status of Cells To Check Genetic Stability of Cells Techniques of Characterization Techniques for Species Verification Chromosome Banding Protocol: Chromosome Banding DNA Content DNA Hybridization Isoenzymology Protocol 2: Isoenzyme Analysis Immunological Tests Techniques to Detect Intra Species Contamination Techniques for Characterizing Cell Types Techniques to Detect Microbial Contamination Preservation of Animal Cell Lines Steps of Cryopreservation Evaluation of Cell Viability Thawing Cell Banks SCALE UP OF ANIMAL CELL CULTURE Practical Considerations during Animal Cell Culture Culture Parameters Medium Non Nutritional Medium Supplements Oxygen Redox Potential Growth Kinetics Culture Vessel and Growth Surfaces Cell Quantification Scale Up Process Scale Up of Monolayer Nunc Cell Factory Multitray Disks, Spirals and Tubes Roller Bottles / Tubes Microcarriers Perfused Monolayer Culture

10 Microencapsulation Scale Up of Suspension Protocol: Suspension Cultures Continuous Culture Mixing and Aeration in Suspension Airlift Fermenters Perfused Suspension Culture Rotating Chambers Fluidized Bed Reactors for Suspension Culture On Line Monitoring of Cell Culture FERMENTORS Continuous Culture The Components of a Fermentor Parts of Fermentors Vessel Peripheral Parts and Accessories Reagent Pumps Medium Feed Pumps and Reservoir Bottles Rotameter / Gas Supply Sampling Device Alternative Vessel Designs Air Lift Fluidized Bed Hollow Fibre In Situ Sterilizable Fermentors Containment Additional Accessories and Peripherals Feed Pumps Exit Gas Analysis Infra-Red Carbon Dioxide Analyzer Paramagnetic Oxygen Analyzer Mass Spectrometer Types of Reactors Mechanically Agitated Stirred Tank Reactors Air-Lift Bioreactor and Modified Air Lift Bioreactors Novel Seesaw Bioreactor Different Types of Fermentor Instrumentation Analogue Controllers Rack System Analogue Controllers Separate Modules in Housings Digital Controllers Embedded Microprocessor Digital Controllers Process Controllers Digital Controllers Direct Computer Control Common Measurement and Control Systems Speed Control

11 Temperature Control Control of Gas Supply Control of Dissolved Oxygen Antifoam Control Transfer of Oxygen Maintenance of Aseptic Conditions HYBRIDOMA TECHNOLOGY AND MONOCLONAL ANTIBODIES Hybridoma Limitation of Traditional Method of Antibody Production Advantages and Disadvantages of Mabs Over Polyclonal Antibodies Types of MAbs Chimeric MAbs Hybridoma Technology The Basis of Hybridoma Technology Production of Monoclonal Antibodies Choice of Fusion Partners Immunization of Mice Making Hybridomas Screening Procedures Long Term Storage Hybridomas from Different Species Production of MAbs through Genetic Engineering Alternative to Hybridoma Production of Human/Humanized Antibodies Application of MAbs Diagnosis Screening and Therapy Monoclonal Antibodies as Enzymes (Abzymes) Vaccine Production Purification and Quantitation of Compounds Raising MAbs Purification and Quantitation Radio Immunoassay (RIA) Cytogenetic Analysis GENETIC MANIPULATION OF CELLS: TRANSGENESIS Why Animal Cells are Chosen for Genetic Manipulation Basics of Genetic Manipulation Isolation of Gene Introduction of Gene into Suitable Vector Introduction of Recombinant Vector into Cells Calcium Phosphate Transfection Lipofection Electroporation Microinjection

12 Retroviral Infection Embryonic Stem Cell Transfer Microprojectile / Biolistic Screening for Recombinant Cells Analysis of Transgene Integration Detection of mrna Expression Assay for Protein Expression Examples of Transgenic Animals Integration of Microinjected Sequences into the Embryo: A Case Study of Mice Genotyping Transgenic Mice by PCR Specific Applications of Transgenic Mice Gene Expression Transgenic Mice as Models for Genetic Engineering Transgenic Swine Production of Sheep Transgenic for Growth Hormone Genes Insertion of Genes Expression of Transgenes Production of Transgenic Cattle by Pronuclear Injection Collection of Embryos Pronuclear Injection Preparation of DNA Culture and Transfer of Embryos Collection and Analysis of Tissue Methods for the Introduction of Recombinant DNA into Chicken Embryos Problems after Developing Transgenic Animals The Nature of Patents APPLICATIONS OF TRANSGENIC ANIMALS Transgenic Animal Technology Genuine Transgenic Animals and their Applications Enhanced and Tissue-Specific Transgene Expression Production of Sheep Transgenic for Growth Hormone Genes Production of Transgenic Cattle by Pronuclear Injection Methods for the Introduction of Recombinant DNA into Chicken Embryos Present States of Art Gene Transfer by Microinjection Other Methods Use for Medical and Therapeutic Purposes Recombinant Protein Production Xenotransplantation Genetic Engineering- Single Genes Gene Mapping Artificial Insemination and Germ Cell Storage Ectogensis Amniocentesis

13 Significance of Amniocentesis Transplantation Cloning Technique Gene Therapy Genetic Counselling In Vitro Fertilization and Embryo Transfer Surrogate Motherhood In Vitro Fertilization and Embryo Transfer for Improved Breeding Programmes Success of Embryo Transplantation Objectives and Applications of ET Superovulation Physiological Basis of Superovulation Factors Which Influence the Superovulatory Response Superovulation using Pmsg Freezing of Embryos Frozen Embryos Embryo Sexing Success in Pre-Determining the Sex Micromanipulation of Embryos Advantages of Cell Manipulation Technique Simple Method of Splitting Embryos Techniques of Nuclear Transplantation Sources of Eggs and Embryos Gene Injection (Introduction of Foreign Cloned Genes) Dense Cytoplasm and Gene Injection Selective Animal Breeding Intensity of Selection and Reproductive Rate Potential Hazards of Artificial Breeding Animals as Tennis Balls? Down on the Farm Time to Decide TISSUE GRAFTING AND ENGINEERING Introduction Historical Perspective Principle Prerequisite for Tissue Engineering Semi-Natural and Natural Substrate Synthesis of Tissue Engineering Scaffolds Cell Sources Assembly Methods Some Examples of Tissue Engineering Use of Biological Film as a Cultured Epidermal Autograft Tissue Engineering Peripheral Nerve Implants

14 Tissue Engineering for Vascular Graft Advantages and Disadvantages of Tissue Engineering Ethical Issues Future Prospects GENE THERAPY How does Gene Therapy Work? Gene Therapy in the Treatment of Cancer Main Approaches of Gene Therapy Gene Therapy and Transfer of Genes Into Cells Viruses used as Gene Therapy Vectors Retroviruses Adenoviruses Adeno-Associated Viruses Herpes Simplex Viruses Current Status of Gene Therapy Research Gene Therapy Factors and Genetic Disease Short-Lived Nature of Gene Therapy Immune Response Problems with Viral Vectors Multigene Disorders Recent Developments in Gene Therapy Subtle Gene Therapy Tackles Blood Disorder DNA Nanoballs Boost Gene Therapy Risks Associated with Gene Therapy Trials Precautions for Gene Therapy REFERENCES SUBJECT INDEX

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16 Animal Cell And Tissue Culture Publisher : Agrobios Publications ISBN : Author : Mathur S Type the URL : Get this ebook