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Cell culture in animal biotechnology

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1. CELL CULTURE IN ANIMAL BIOTECHNOLOGY Name :Priya Yadav Sumitted to : Dr. Namarata Ma’am 2. INTRODUCTION Cell culture refers to the process by which cells are grown…
  • 1. CELL CULTURE IN ANIMAL BIOTECHNOLOGY Name :Priya Yadav Sumitted to : Dr. Namarata Ma’am
  • 2. INTRODUCTION Cell culture refers to the process by which cells are grown in a controlled artificial environment. Cells can be maintained in vitro. The cells may be removed directly or by mechanical or enzymatic action. Examples : fibroblast, lymphocytes, cells from cardiac and skeletal tissues, cells from liver, breast, skin, and kidney and different types of tumor cells.
  • 3. HISTORY 1878: Claude Bernard proposed that physiological systems of an organism can be maintained in a living system after the death of an organism. 1885: Roux maintained embryonic chick cells in a saline culture. 1897: Loeb demonstrated the survival of cells isolated from blood and connective tissue in serum and plasma. 1903: Jolly observed cell division of salamander leucocytes in vitro. 1907: Harrison cultivated frog nerve cells in a lymph clot held by the ‘hanging drop’ method
  • 4. 1910: Burrows succeeded in long-term cultivation of chicken embryo cell in plasma clots. He made detailed observation of mitosis. 1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth. 1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods. 1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells. 1923: Carrel and Baker developed ‘Carrel’ or T-flask as the first specifically
  • 5. 1982: Human insulin became the first recombinant protein to be licensed as a therapeutic agent. 1985: Human growth hormones produced from recombinant bacteria was accepted for therapeutic use. 1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available. 1989: Recombinant erythropoietin in trial. 1990: Recombinant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF, EGF, mAbs, IL-2)
  • 6. REQUIREMENTS Equipments: Sterile Work Area, Incubation Facilities , Refrigerators and Freezer , Microscopes , Tissue Culture Ware , Washing Up and Sterilizing Facilities , Liquid Nitrogen Deep Freezer , Water Still or Reverse Osmosis Apparatus , Filter Sterilization. Environmental requirements: optimum ph approx.7-7.5 , temperature 37⁰ C, osmotic pressure 300mOsm , medium acidic buffer , growth factors such as cysteine and tyrosine ,glutamine etc ,antibiotics and antimycotics. Culture media: mixture of Organic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace elements,vitamins, cofactors, and ions. Glucose is added as energy source.
  • 7. Natural Media: 1. Coagulant, such as plasma clots. 2. Biological fluids such as serum 3. Tissue extracts for example Embryo extracts Synthetic Media: Synthetic media are prepared artificially by adding several organic and inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, co-factors, etc. 1. Serum containing media (media containing serum) 2. Serum- free media (media without serum). Ex. minimal essential medium (MEM), RPMI 1640 medium, CMRL 1066, F12, etc.
  • 8. PROCESS
  • 9. TYPES OF ANIMAL CELL CULTURE Primary culture When cells are surgically removed from an organism and placed into a suitable culture environment, they will attach, divide and grow.  Adherent cells : These cells are anchorage dependent and propagate as a monolayer. Needed to be attached to a solid or semi-solid substrate for proliferation. These adhere to the culture vessel with the use of an extracellular matrix which is generally derived from tissues of organs that are immobile and embedded in a network of connective tissue. EX. Fibroblasts and epithelial cells are of such types. Suspension cells : These do not attach to the surface of the culture vessels. These cells are also called anchorage independent or non-adherent cells which can be grown floating in the culture medium . EX. Hematopoietic stem cells and tumor cells can be grown in suspension
  • 10. Secondary culture: When a primary culture is sub-cultured, it is known as secondary culture or cell line or sub-clone. The process involves removing the growth media and disassociating the adhered cells (usually enzymatically). Sub-culturing of primary cells to different divisions leads to the generation of cell lines. On the basis of the life span of culture, the cell lines are categorized into two types: Finite cell lines: The cell lines which go through a limited number of cell division having a limited life span are known as finite cell lines. Continuous cell lines: When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line.
  • 11. CHARACTERISTICS Cell Culture Systems:  Ability of the cells to either grow attached to a glass or treated plastic substrate, called as monolayer culture systems .  Floating free in the culture medium called as Suspension Culture Systems. Types of Cells: Epithelial Lymphoblast like  Fibroblast like
  • 12. Functional Characteristics: Based on their origin and adaptation Markers used can be morphological or biochemical Some cell lines will eventually stop dividing and show signs of aging in artificial environment . These lines are called Finite lines. Lines which become immortal can continue to divide indefinitely and are called Continuous cell lines. When a “normal” finite cell line becomes immortal, it undergoes a fundamental irreversible change or “transformation”.
  • 13. Cells that have the normal number of chromosomes are called Diploid cells. Cells having other than the normal number are Aneuploid. If cells form tumours when they are injected into animals, they are considered to be Neo-plastically Transformed.
  • 14. ADVANTAGES  Controlled physiochemical environment  Controlled and defined physiological conditions  Homogeneity of cell types (achieved through serial passages) Legal, moral and ethical questions of animal experimentation are avoided. Economical
  • 15. DISADVANTAGES  Expertise is needed. Ten times more expensive.  Unstable aneuploid chromosome constitution.
  • 16. APPLICATIONS Model system for study Toxicity testing Cancer research Virology Cell based manufacturing Genetic counselling and engineering Drug screening and development Gene therapy
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