I am working on normal primary oral keratinocyte cells. The cells have been isolated from enzymatic digested explant method. Cells were healthy after first split (passage=1), however, after second split (passage=2) cells start to form vacuole-like shape. Cell death and slow growth rate are also started. The same cell-characteristic was also observed in my previous primary cell line (after passage 3). Images of p=1 and p=2 are attached. Please suggest how to resolve the problem.
Hey I suspect these cell were under stress ( Coz Vacuoles formation is the sign of stress). Check your culture media pH. Try with freashly prepared media and avoid frequent thawing of media. Also backcheck your media composition, try with some specific media recommended for Keratinocytes.
I used serum free keratinocyte growth medium supplemented with EGF, insulin and bovine pituitary extract. I also used 5% FBS and 1% antibiotic-antimycotic solution. In my previous work i used 10% FBS instead of 5%. In that case cells were continued upto passage 3.
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