Animal Cell Culture

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Cell Cultures/ Dispersion Cultures

• Types

• Primary cell cultures

• Diploid cell cultures

• Continuous cell lines

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Primary Cell Culture

• Sterilization of Cubicle

• Preparation of Media• Preparation of

dispersion solution• Preparation of

Monolayers

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Sterilisation of Cubicle

• Fumigation• 35 ml of formaline and 17.5 g of potassium

permanganate per 100 cubic feet of air space

• Keep the cubicle closed for 65 hours

• Working Surface Sterilization• Mop with 5% phenyl/ 1/40 solution of dettol

• Use 1 % Copper sulphate for fungal disinfection

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Cell Culture Media

• Balanced Salt Solution (BSS)

• Glucose

• Amino acids

• Vitamins

• Sodium bicarbonate

• Phenol red

• Fetal Calf Serum

Institute of Animal Health and Veterinary Biologicals

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Culture Media Prepare stock antibiotic solution

Weigh the required amount of Ready made Balanced Salt Medium and

add sterile water

Add the required amount of antibiotic stock solution into BSS

Adjust the pH of the solution using 2.8% / 7.5% Sodium bicarbonate

or 0.1 N Hydrochloric acid to 7.4

Sterilize using 0.2 Seitz filter applying positive pressure.

Add sterile heat inactivated fetal calf serum to make a final

concentration of 15%

Store at 4o C.

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Dispersion solution

• Weigh Calcium Magnesium free Buffer premix and add sterile water

• Add 0.1 % w/v Trypsin powder

• Adjust pH to 7.4 to 7.6

• Sterilize by Filtration through Seitz filter

• Store at - 20o C

Primary Cell Culture

Chicken Embryo Fibroblast

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Materials Ten to Eleven days Embryonated Chicken Eggs

Forceps, Scissors, Rubber bulbs

Pipettes, Beakers, Centrifuge tubes, Flasks, Petri dishes, Trypsinisation flask

Seitz filter assembly, Vacuum/air pump

Mono-pan analytical balance

pH meter

Magnetic stirrer, Teflon coated magnetic stirrer, Cyclo-mixer

Refrigerated Centrifuge

Laminar Air Flow Apparatus

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Preparation of Chicken Embryo Fibroblast Preparation of Chicken Embryo Fibroblast MonolayerMonolayer

1. Sterilize Outer shell of the Eggs 2. Remove the Shell, Shell membrane and CAM3. Wash embryo with BSS4. Remove Head, Limbs and Internal Organs5. Wash several times with BSS6. Cut into small pieces of ~ 1mm thickness7. Wash in BSS8. Trypsinize /.9. Filter with a cheese cloth10. Centrifuge and pack cells11. Resuspend in growth medium12. Repeat step 10 and 11, two more times13. Count the cells and adjust to a concentration of 10 6 cells per ml14. Seed Culture tubes/ flasks/ Bottles and Incubate at 37 o C for 48 to 72

hours.

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procedure

• Opening of egg through a circular incision around the airsac

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• Transfer the live embryo to a petri dish containing media

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• Collect more embryos

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• Remove the appendages and viscera

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• Collect the remaining portion

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• Cut the embryo in to small parts (about 1 mm)

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• Trypsinise in a flask

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• Keep in magnetic stirrer for about 30 minutes

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• Centrifuge the cell suspension

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• Wash the cells 3 more times.

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• Mix well in cyclo mixer

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• Incubate for about 30 min at 4oc

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• Dispense in cell culture bottles

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• Incubate at 37oc in a CO2 incubator

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