Subculturing primary Keratinocytes - (Jun/05/2001 )

Hi everybody,

I'm trying to isolate and culture Keratinocytes from human Skin. Isolation and culturing works well, but I've got problems subculturing them. I'm working with a Trypsin/EDTA solution in PBS without Calcium an Magnesium. Sometimes subculturing works and sometimes not. I have also problems with Fibroblasts overgrowing my Keratinocytes.I use a serumfree Medium caled EpiLife from Cascade Inc.

If somebody has experience with Keratinocyte Cultures, contact me.

Your problem may be that the keratinocytes are getting confluent.Human keratinocytes must be passaged before they get confluent and differentiate.We use a low concentration of trypsin (0.03%) with 0.01% EDTA.Also, you are only going to be able to pass a few times before the senesce.

I hope this helps.

Mitchell Denning

i used trypsin-EDTA 0.05% and versene to detach cells. try to subculture when confluency is 50-60%. trypsinized mildly, but use versene to wash it twice prior to trypsinize...time to incubation is another problem...cos concentration of trypsin-EDTA and time to incubate it in trypsin containing media will kill or lead to mitosis breakage.