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Subculturing primary Keratinocytes


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#1 anonymous

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Posted 05 June 2001 - 09:00 PM

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.

#2 anonymous

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Posted 08 June 2001 - 09:00 PM

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


#3 sycay

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Posted 25 December 2004 - 11:03 PM

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. :)




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