Cellculure

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animal cell culture techniques

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Cellculure

  1. 1. Animal Cell Culture ubio www.ubio.in
  2. 2. Cell Cultures/ Dispersion Cultures <ul><li>Types </li></ul><ul><ul><ul><li>Primary cell cultures </li></ul></ul></ul><ul><ul><ul><li>Diploid cell cultures </li></ul></ul></ul><ul><ul><ul><li>Continuous cell lines </li></ul></ul></ul>ubio www.ubio.in
  3. 3. Primary Cell Culture <ul><li>Sterilization of Cubicle </li></ul><ul><li>Preparation of Media </li></ul><ul><li>Preparation of dispersion solution </li></ul><ul><li>Preparation of Monolayers </li></ul>
  4. 4. Sterilisation of Cubicle <ul><li>Fumigation </li></ul><ul><ul><ul><li>35 ml of formaline and 17.5 g of potassium permanganate per 100 cubic feet of air space </li></ul></ul></ul><ul><ul><ul><li>Keep the cubicle closed for 65 hours </li></ul></ul></ul><ul><li>Working Surface Sterilization </li></ul><ul><ul><ul><li>Mop with 5% phenyl/ 1/40 solution of dettol </li></ul></ul></ul><ul><ul><ul><li>Use 1 % Copper sulphate for fungal disinfection </li></ul></ul></ul>
  5. 5. Cell Culture Media <ul><li>Balanced Salt Solution (BSS) </li></ul><ul><li>Glucose </li></ul><ul><li>Amino acids </li></ul><ul><li>Vitamins </li></ul><ul><li>Sodium bicarbonate </li></ul><ul><li>Phenol red </li></ul><ul><li>Fetal Calf Serum </li></ul>
  6. 6. Culture Media <ul><li>Prepare stock antibiotic solution </li></ul><ul><li>Weigh the required amount of Ready made Balanced Salt Medium and add sterile water </li></ul><ul><li>Add the required amount of antibiotic stock solution into BSS </li></ul><ul><li>Adjust the pH of the solution using 2.8% / 7.5% Sodium bicarbonate or 0.1 N Hydrochloric acid to 7.4 </li></ul><ul><li>Sterilize using 0.2  Seitz filter applying positive pressure. </li></ul><ul><li>Add sterile heat inactivated fetal calf serum to make a final concentration of 15% </li></ul><ul><li>Store at 4 o C. </li></ul>Institute of Animal Health and Veterinary Biologicals
  7. 7. Dispersion solution <ul><li>Weigh Calcium Magnesium free Buffer premix and add sterile water </li></ul><ul><li>Add 0.1 % w/v Trypsin powder </li></ul><ul><li>Adjust pH to 7.4 to 7.6 </li></ul><ul><li>Sterilize by Filtration through Seitz filter </li></ul><ul><li>Store at - 20 o C </li></ul>
  8. 8. Primary Cell Culture Chicken Embryo Fibroblast
  9. 9. Materials <ul><li>Ten to Eleven days Embryonated Chicken Eggs </li></ul><ul><li>Forceps, Scissors, Rubber bulbs </li></ul><ul><li>Pipettes, Beakers, Centrifuge tubes, Flasks, Petri dishes, Trypsinisation flask </li></ul><ul><li>Seitz filter assembly, Vacuum/air pump </li></ul><ul><li>Mono-pan analytical balance </li></ul><ul><li>pH meter </li></ul><ul><li>Magnetic stirrer, Teflon coated magnetic stirrer, Cyclo-mixer </li></ul><ul><li>Refrigerated Centrifuge </li></ul><ul><li>Laminar Air Flow Apparatus </li></ul>
  10. 10. Preparation of Chicken Embryo Fibroblast Monolayer <ul><li>1. Sterilize Outer shell of the Eggs </li></ul><ul><li>2. Remove the Shell, Shell membrane and CAM </li></ul><ul><li>3. Wash embryo with BSS </li></ul><ul><li>4. Remove Head, Limbs and Internal Organs </li></ul><ul><li>5. Wash several times with BSS </li></ul><ul><li>6. Cut into small pieces of ~ 1mm thickness </li></ul><ul><li>7. Wash in BSS </li></ul><ul><li>8. Trypsinize /. </li></ul><ul><li>9. Filter with a cheese cloth </li></ul><ul><li>10. Centrifuge and pack cells </li></ul><ul><li>11. Resuspend in growth medium </li></ul><ul><li>12. Repeat step 10 and 11, two more times </li></ul><ul><li>13. Count the cells and adjust to a concentration of 10 6 cells per ml </li></ul><ul><li>14. Seed Culture tubes/ flasks/ Bottles and Incubate at 37 o C for 48 to 72 hours. </li></ul>
  11. 11. procedure <ul><li>Opening of egg through a circular incision around the airsac </li></ul>
  12. 12. <ul><li>Transfer the live embryo to a petri dish containing media </li></ul>
  13. 13. <ul><li>Collect more embryos </li></ul>
  14. 14. <ul><li>Remove the appendages and viscera </li></ul>
  15. 15. <ul><li>Collect the remaining portion </li></ul>
  16. 16. <ul><li>Cut the embryo in to small parts (about 1 mm) </li></ul>
  17. 17. <ul><li>Trypsinise in a flask </li></ul>
  18. 18. <ul><li>Keep in magnetic stirrer for about 30 minutes </li></ul>
  19. 19. <ul><li>Centrifuge the cell suspension </li></ul>
  20. 20.
  21. 21. <ul><li>Wash the cells 3 more times. </li></ul>
  22. 22. <ul><li>Mix well in cyclo mixer </li></ul>
  23. 23. <ul><li>Incubate for about 30 min at 4 o c </li></ul>
  24. 24. <ul><li>Dispense in cell culture bottles </li></ul>
  25. 25. <ul><li>Incubate at 37 o c in a CO 2 incubator </li></ul>
  26. 26.
  27. 27. <ul><li>THANK YOU </li></ul>

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