Keratinocy grows together with fibroblast. I am using keratinocyte growth media. Is there a way to remove fibroblast?
Dear Seong Jun Lee,
By differential trypsinization you can remove fibroblast from a keratinocyte -feeder co-culture. First you remove the feeders with the help of EDTA. The EDTA conc. depends on type of feeder cells (fibroblast) used for co-culture. Generally for Mouse embryonic fibroblast 3T3 cells the EDTA conc. should be 0.03%.
Hope this will work for you or you refer http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122056.
Yes. Agreed. Fibroblasts are much more readily removed by trypsinization in comparison to keratinocytes. A gentle trypsinization will remove fibroblasts but will leave keratinocytes behind.
Differential trypsinization plus serum-free culture condition. I recommend you to use commercial available keratinocyte medium which is a serum-free medium. in that culture condition, fibroblasts are very skinny and easily be removed by differential trypsinization. on the other hand, serum-free culture condition inhibit fibroblasts grow. Good luck!
Thank you everyone for answer me.
I cultivate keratinocyte under serum-free conditions. Fibroblast grows even when subculture is carried out. Does the BPE included in the medium help fibroblast growth? How long is the trypsin treatment time when subculturing? Do long-time treatment of trypsin to damage keratinocyte? Keratinocyte is not well detach from the culture plate.
Keratinocyte serum-free medium with BPE does not support fibroblast growth. but give support to keratinocytes. normally with feeder system the trypsin conc is about .2 to .4 % but in feeder less (serum-free medium) condition. the trypsin conc. should be about 3-4 times diluted. because strong trypsin will effect the keratinocytes output.
For detaching keratinocyte from in feeder-less system use only diluted trypsin and you have to keep the culture for longer period at 37 degree C with intermittent observation under microscope.
Hope this will help you. All the best.
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