I did cell culture of liver tissue isolated from 14 days old chicken embryo and the method I followed was the same as like chicken embryo fibroblast cell culture. But I am facing problems after the first passage i.e after trypsinization. all cells are dead after 24hrs of passaging. I tried 0.25% and 0.125% trypisn EDTA for trypsinization. The medium I used was M-199 with 10% FBS and antibiotic added. thank you
You may miss something Check out Protocol again, although may be cell culture with low efficiency to produce glucose.did you used humidified incubator (95% air and 5% CFO2, 38.5C) ?
culture of hepatocytes from chicken embryos protocol :
1. Submerge in 70% alcohol and 5 min six or more embryonated chicken eggs which have been incubated for 12 to 18 days.
2. Place the surface sterilized eggs, large end up, on a clean plastic egg flat to let the alcohol evaporate (about 3 min).
3. Crack the shell on the large end and remove the embryo to a sterile Petri dish, using two pairs of forceps previously dipped in alcohol.
4. Tease the abdominal wall open, excise the liver, and place the liver in a separate dish containing buffered calcium- and magnesium-free saline (CMF-saline).
5. After all livers have been removed and submerged in CMF-saline, tease away the gall bladders and transfer the liver tissues to clean CMF-saline.
6. The liver tissues are finally transferred to a sterile beaker containing 20 ml of prewarmed CMF-saline (38.5 C) with 0.1% collagenase (Type 1A, from Clostridium histolyticum, Sigma Chemical Co., sterilized by filtering through 0.2 um membrane) and digested for 10 min with stirring.
7. Stop the digestion by mixing with 20 ml of cold (4 C) Medium 199, and the mixture poured over sterile 4 layers of cheese cloth.
8. Collect the freed cell by centrifugation at 500 g for 5 min and resuspend the cell pellete 8 ml of CMF-saline.
9. Lay 4 ml cell suspension atop 2 ml of Histopaque 1077 (Sigma Chemical Co., St. Louis, MO, prewarmed to room temperature) and centrifuge at 700 g for 15 min to precipitate the red blood cells.
10. Transfer the cells (mainly liver parenchymal cells) at the interface to a sterile glass test tube and washed twice in CMF by centrifugation for 5 min at 500 g.
11. Resuspend the cells in Medium 199 (or other medium of choice) amended with 10% head inactivated fetal bovine serum and antibiotics (100 IU penicillin, 100 ug sterptomycin and 0.25 ug amphrotericin B/ ml, final concentration).
12. Determine the viable cell concentration by mixing 30 ul of cell with 30 ul of 0.4% trypan blue in PBS, transferring the mixture to the hemocytometer, leaving for 1 to 2 min and counting the number of non stained cells.
13. Adjusted the cell to a desirable concentration and dispense into culture dishes of choice.
Thank you so much Alaa Abed...... I used humidified incubator (95% air and 5% CFO2, 38.5C) chamber for incubation. but instead of Collagenase i used Trypsin for dis-aggregation of tissue.
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