Cellculure

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Information about Cellculure

Published on March 11, 2008

Author: bishorvi

Source: slideshare.net

Description

animal cell culture techniques

Animal Cell Culture ubio www.ubio.in

Cell Cultures/ Dispersion Cultures Types Primary cell cultures Diploid cell cultures Continuous cell lines ubio www.ubio.in

Types

Primary cell cultures

Diploid cell cultures

Continuous cell lines

Primary Cell Culture Sterilization of Cubicle Preparation of Media Preparation of dispersion solution Preparation of Monolayers

Sterilization of Cubicle

Preparation of Media

Preparation of dispersion solution

Preparation of Monolayers

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

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

Cell Culture Media Balanced Salt Solution (BSS) Glucose Amino acids Vitamins Sodium bicarbonate Phenol red Fetal Calf Serum

Balanced Salt Solution (BSS)

Glucose

Amino acids

Vitamins

Sodium bicarbonate

Phenol red

Fetal Calf Serum

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 4 o C. Institute of Animal Health and Veterinary Biologicals

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 4 o C.

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 - 20 o C

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 - 20 o C

Primary Cell Culture Chicken Embryo Fibroblast

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

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

Preparation of Chicken Embryo Fibroblast Monolayer 1. Sterilize Outer shell of the Eggs 2. Remove the Shell, Shell membrane and CAM 3. Wash embryo with BSS 4. Remove Head, Limbs and Internal Organs 5. Wash several times with BSS 6. Cut into small pieces of ~ 1mm thickness 7. Wash in BSS 8. Trypsinize /. 9. Filter with a cheese cloth 10. Centrifuge and pack cells 11. Resuspend in growth medium 12. Repeat step 10 and 11, two more times 13. Count the cells and adjust to a concentration of 10 6 cells per ml 14. Seed Culture tubes/ flasks/ Bottles and Incubate at 37 o C for 48 to 72 hours.

1. Sterilize Outer shell of the Eggs

2. Remove the Shell, Shell membrane and CAM

3. Wash embryo with BSS

4. Remove Head, Limbs and Internal Organs

5. Wash several times with BSS

6. Cut into small pieces of ~ 1mm thickness

7. Wash in BSS

8. Trypsinize /.

9. Filter with a cheese cloth

10. Centrifuge and pack cells

11. Resuspend in growth medium

12. Repeat step 10 and 11, two more times

13. Count the cells and adjust to a concentration of 10 6 cells per ml

14. Seed Culture tubes/ flasks/ Bottles and Incubate at 37 o C for 48 to 72 hours.

procedure Opening of egg through a circular incision around the airsac

Opening of egg through a circular incision around the airsac

Transfer the live embryo to a petri dish containing media

Transfer the live embryo to a petri dish containing media

Collect more embryos

Collect more embryos

Remove the appendages and viscera

Remove the appendages and viscera

Collect the remaining portion

Collect the remaining portion

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

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

Trypsinise in a flask

Trypsinise in a flask

Keep in magnetic stirrer for about 30 minutes

Keep in magnetic stirrer for about 30 minutes

Centrifuge the cell suspension

Centrifuge the cell suspension



Wash the cells 3 more times.

Wash the cells 3 more times.

Mix well in cyclo mixer

Mix well in cyclo mixer

Incubate for about 30 min at 4 o c

Incubate for about 30 min at 4 o c

Dispense in cell culture bottles

Dispense in cell culture bottles

Incubate at 37 o c in a CO 2 incubator

Incubate at 37 o c in a CO 2 incubator



THANK YOU

THANK YOU

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