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Animal biotechnology used equipment and media used for animal cell culture technology

Molecular biology and recombinant DNA technology- Animal biotechnology used equipment and media used for cell culture technology

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ANIMAL BIOTECHNOLOGY – EQUIPMENT AND MEDIA USED FOR ANIMAL CULTURE
VIVEKANANDHA ARTS AND SCIENCE COLLEGE FOR WOMAN Veerachipalayam-637 303,Sankagiri,Salem Dt, Tamilnadu,India. Affiliated to Periyar University Salem ; recognised under section 2(f)&12(b) of the UGC act,1956 DEPARTMENT OF MICROBIOLOGY TITLE : ANIMAL BIOTECHNOLOGY – EQUIPMENT AND MEDIA USED FOR ANIMAL CELL CULTURE SUBJECT INCHARGE : Mythili Dr. R, Head Of the Department Department of Microbiology VIAAS , Sankagiri . SUBMITTED BY : Nandhini Vengadachalam II M.sc Microbiology Department of Microbiology VIAAS , Sankagiri . SUBJECT : MOLECULAR BIOLOGY AND RECOMBINANT DNA TECHNOLOGY
CONTENT Introduction Equipment Media used animal culture Animal cell culture Application Advantages Disadvantages Current animal technology Reference
INTRODUCTION Animal biotechnology is the combination of science and engineering which is very useful to transform living organisms. The main objective of animal biotechnology is to make products, to improve animals, and to develop microorganisms for specific agricultural uses. Creating transgenic animals is the best example of animal biotechnology.
ANIMAL CELL CULTURE Father of animal cell culture is Ross . G Harrison – 1907 Achieve - first successfully frog nerve tissue outside body (in vitro ) animal cells could survive and grow under laboratory conditions . Roux in 1885 for the first time maintained embryonic chick cells in a cell culture. The culture of animal cells and tissues is a generally and widely used technique that involves isolation of cells, tissues and organs from animals and growing them in an in vitro or artificial environment. The term culture means to keep alive and grow in an appropriate medium that simulates the natural conditions.
ANIMAL CELL CULTURE HISTORY 1885 Maintained embryonic chick cells in a saline culture Roux  1897 Demonstrated the survival of cells isolated from blood and connective tissue in serum and plasma Loeb  1907 Cultivated frog nerve cells in a lymph clot held by the “hanging drop” method and observed the growth of nerve fibers in vitro for several weeks Harrisone 1911 First liquid media consisted of sea water, serum, embryo extract, salts and peptones Lewis and Lewwis 1916 Proteolytic enzyme trypsin for the subculture of adherent cells Rousand Jones  1923 T-flask as the first specifically designed cell culture vessel Carrel and Baker
1948 Isolated mouse l fibroblasts which formed clones from single cells Earle  1949 Polio virus could be grown on human embryonic cells in culture Enders  1952 Continuous cell line from a human cervical carcinoma known as hela (helen lane) cells Gey 1955 Nutrient requirements Eagle 1964 Hat medium for cell selection Littlefield 1975 First Hybridoma capable of secreting a monoclonal antibody Koehler and Milstein
ANIMAL CELL CULTURE TYPES
 Primary cell culture :  Separated by enzymatic or mechanical method  Separated cell directly from the parent tissue (kidney, liver, heart etc.)  Maintaining grow of the cell culture media using plastic containers  Cell two types classified as ,
Primary cell culture ,
Secondary cell culture :  When primary cell is sub cultured then it is secondary cell Sub culture : Transfer of the cell them one culture vessel to another. Method : Remove growth medium Detatch adherent cells(enzymatic method) Why required : To provide fresh nutrients To provide more space
CELL LINE
ANIMAL CELL CULTURE PROCEDURE
EQUIPMENT REQUIRED ANIMAL CELL CULTURE Laminar Air Flow Cabinet CO Incubator ₂ Inverted Microscope Autoclave Centrifuge Water Bath Micropipettes Refrigerator and Deep Freezer Liquid Nitrogen Storage Tank pH Meter Filtration Unit Culture Flasks and plates
LAMINAR AIR FLOW  There are two types of laminar flow hoods, vertical and horizontal. The vertical hood, also well-known as a biology safety cabinet.  Both types of hoods have continuous displacement of air that passes through a HEPA (high efficiency particle) filter use for the purpose of removes particulates from the air.  The hoods are equipped with a short-wave UV light that can be turned on for a few minutes to sterilize the surfaces of the hood, but be aware that only exposed surfaces will be accessible to the UV light.  Do not put your hands or face near the hood when the UV light is on as the short wave light can cause skin and eye damage. The hoods should be turned on about 10 – 20 minutes before being used .
CO2 INCUBATOR CO incubator maintains the optimum temperature, humidity, ₂ and carbon dioxide concentration required for animal cell growth. Uses Maintains 37°C temperature. Maintains 5% CO . ₂ Maintains 95% relative humidity. Supports growth of mammalian cells. Procedure Set temperature to 37°C. Set CO concentration to 5%. ₂ Fill the water tray with sterile distilled water. Place culture flasks inside.  Minimize door opening to avoid temperature fluctuations.
INVERTED MICROSCOPE An inverted microscope has the objective lens below the stage, allowing observation of cells growing in culture vessels. Uses Observe live cells. Check cell morphology. Detect contamination. Monitor cell confluency and cell division. Procedure Place the culture flask on the stage. Focus using low power, then high power. Observe cell shape and growth.  Record observations.
AUTO CLAVE An autoclave sterilizes equipment using steam under high pressure. Uses Sterilizes media. Sterilizes glassware and instruments. Decontaminates laboratory waste. Procedure Fill the autoclave with water. Load materials loosely. Sterilize at 121°C, 15 psi, for 15–20 minutes.  Allow pressure to return to zero before opening.
CENTRIFUGE A centrifuge separates cells from liquids using centrifugal force. Uses Collect cells. Wash cells. Remove supernatant. Prepare cell pellets. Procedure Transfer cell suspension to centrifuge tubes. Balance tubes. Spin at required speed (usually 1000–1500 rpm for 5–10 min).  Remove supernatant carefully.
WATER BATH A water bath maintains samples at a constant temperature. Uses Warm culture media. Thaw frozen cells. Warm reagents. Procedure Set temperature to 37°C. Immerse the sample without submerging the cap. Remove immediately after thawing.  Wipe with 70% ethanol before use.
MICROPIPETTES Micropipettes accurately measure and transfer small liquid volumes. Uses Transfer media. Add serum. Pipette cell suspension. Prepare reagents. Procedure Attach a sterile tip. Set the required volume. Aspirate liquid. Dispense slowly.  Discard the tip safely.
REFRIGERATOR (4°C) AND DEEP FREEZER (-20°C/-80°C) Cold storage equipment for biological materials. Uses 4°C: Store media and reagents. -20°C: Store enzymes and chemicals. -80°C: Store cell stocks and biological samples. Procedure Label all materials. Store at the correct temperature.  Avoid repeated freeze–thaw cycles
LIQUID NITROGEN STORAGE TANK A cryogenic storage system containing liquid nitrogen at −196°C. Uses Long-term preservation of cell lines. Maintains cell viability. Procedure Suspend cells in cryoprotective medium (usually with DMSO). Freeze gradually. Transfer cryovials to liquid nitrogen.  Retrieve only when needed.
CULTURE FLASK AND PLATES Sterile plastic or glass vessels used to grow animal cells. Uses Cell attachment. Cell growth. Cell maintenance. Experimental studies. Procedure Add sterile culture medium. Seed cells into the flask or plate. Incubate at 37°C with 5% CO . ₂ Observe growth regularly using an inverted microscope.  Replace medium and subculture when cells reach 80–90% confluency
MEDIA USED FOR ANIMAL CELL CULTURE  Basic Components in the Culture Media Most animal cell culture media are generally having following 10 basic components and they are as follows,  Energy sources: Glucose, Fructose, Amino acids, Nitrogen sources: Amino acids.  The various types of media used for tissue culture may be grouped into two broad categories:  1) Natural media  2) Artificial media.
Basal Medium Eagle (BME) – Developer: Harry Eagle (1955) Source: Modified from Eagle’s amino acid studies. Contains: Essential amino acids, Vitamins,Glucose,Mineral salts Uses:Fibroblasts,Kidney cells ,Primary cell cultures Minimum Essential Medium (MEM) Developer: Harry Eagle (1959) Source: Improved form of BME. Contains: Essential amino acids,Vitamins,Glucose,Phenol red,Sodium bicarbonate Uses: Mammalian cell lines, Vaccine production,General laboratory culture
Dulbecco’s Modified Eagle Medium (DMEM) Developer: Renato Dulbecco source : Source: Modified MEM. Contains: High glucose (4.5 g/L), Amino acids, Vitamins, Sodium pyruvate-glutamine Uses: Stem cells,HEK-293 cells,Fibroblasts,Cancer cell line line Medium 199 (M199) Developer: Morgan, Morton & Parker Source: Developed for nutritional studies. Contains: More than 60 nutrients, Amino acids,Vitamins,Nucleotides Uham’Primary cultures, Vaccine production, Chicken embryo fibroblasts
Ham’s F-10 Medium Developer: Richard Ham  Source: Serum-free culture studies. Uses: CHO cells, Fibroblasts, Mammalian cells Ham’s F-12 Medium - Source: Modified Ham’s F-10. Contains: Trace elements,Zinc,Copper, Putrescine Uses: CHO cells, Hybridoma cells, Serum-free culture H. Iscove’s Modified Dulbecco’s Medium (IMDM) - Developer: Norman Iscove’s Source: Improved DMEM. Uses: Bone marrow cells, Lymphocytes, Hybridoma cell , Hematopoietic cells
Leibovitz’s L-15 Medium - Developer: Allan Leibovitz Source: Designed for CO -independent culture. ₂ Contains: Galactose, Pyruvate,Free-base amino acids  Uses: Cells cultured outside CO incubators, Transport of cells ₂ ANTIBIOTICS USED FOR ANIMAL CELL CULTURE : Peninicillin G - Penicillium chrysogenum (fungus)Mainly Gram-positive bacteria100 Ug/Ml inhibits bacterial cell wall synthesis Streptomycin - Streptomyces griseus (antinomies)Mainly Gram-negative bacteria100 µg/mLInhibits bacterial protein synthesis Gentamicin - Micromonospora purpureaBroad-spectrum bacteria50 µg/mLControls both Gram- positive and Gram-negative bacteria Amphotericin B - (antifungal) Streptomyces nodosus,Yeasts and fungi,0.25–2.5 µg/mL Prevents fungal contamination , Kanamycin- Streptomyces analytics , Broad-spectrum bacteria , 50–100 µg/mL  Inhibits protein synthesis, Neomycin -Streptomyces fradiae
APPLICATION Vaccine production – Used to produce vaccines such as polio, rabies, and influenza vaccines. Monoclonal antibody production – Used to produce antibodies for disease diagnosis and treatment. Drug testing – Used to test the safety and effectiveness of new medicines. Cancer research – Helps scientists study cancer cells and develop new treatments. Gene therapy – Used to study and treat genetic diseases by introducing healthy genes. Stem cell research – Helps develop new treatments for damaged tissues and organs. Tissue engineering – Used to grow artificial skin, bone, cartilage, and other tissues. Recombinant protein production – Used to produce medicines such as insulin and growth hormone. Virus research – Used to study viruses and develop antiviral drugs.  Basic research – Used to study cell growth, cell division, genetics, and cell functions.
ADVANTAGE Provides a controlled environment for cell growth. Reduces the use of experimental animals. Produces reproducible and reliable results. Enables large-scale production of vaccines and therapeutic proteins. Supports genetic engineering and recombinant DNA technology. Useful for disease diagnosis and drug screening. Allows continuous maintenance of cell lines. Facilitates stem cell and regenerative medicine research. Saves time compared with whole-animal experiments.  Essential for modern biomedical and pharmaceutical research.
DISADVANTAGE High cost of equipment, media, and reagents. Requires strict aseptic techniques to prevent contamination. Skilled personnel are needed to maintain cultures. Cell lines may undergo genetic or phenotypic changes after repeated passages. Some primary cells have a limited lifespan in culture. Risk of contamination by bacteria, fungi, viruses, or mycoplasma. Specialized laboratory facilities are required. Mammalian cells grow more slowly than microbial cells. Ethical concerns may arise regarding the use of animal tissues and serum.  Continuous monitoring and maintenance increase operational costs.
CURRENT ANIMAL CELL CULTURE Vaccine production – Used to make vaccines (polio, rabies, influenza, COVID-19). Medicine production – Used to produce medicines such as insulin and monoclonal antibodies. Cancer research – Helps study cancer and develop new treatments. Drug testing – Tests whether new medicines are safe and effective. Stem cell research – Used to repair damaged tissues and organs. Gene therapy – Helps treat genetic diseases by replacing defective genes. Artificial organs and tissues – Used to grow skin, cartilage, and other tissues for transplantation. Virus research – Used to study viruses and develop antiviral medicines. Cultured meat (Lab-grown meat) – Animal cells are used to produce meat without raising or slaughtering animals.  Veterinary research – Helps improve animal health and develop animal vaccines.
REFERENCE Butler M. Animal Cell Culture and Technology.  Glick BR & Pasternak JJ. Molecular Biotechnology: Principles and Applications of Recombinant DNA.  Freshney RI (2016). “Culture of Animal Cells: Principles and Practice.  "Advances in Bioscience and Biotechnology  Received 11 February 2012; revised 22 March 2012; accepted 9 April 2012
Animal biotechnology used equipment and media used for animal cell culture technology