Problems with human keratinocytes - (Apr/06/2010 )
I am new to human cell culture and try to establish primary cell culture from 4mm human forearm skin biopsy. However I experience a few major problems. First the cells grow very slowly, the second after a week fibroblast contamination appear. Could you let me know what I am doing wrong?
When I receive the tissue I trypsinise it for 2 hr at 37C, then using forceps separate dermis, wash in Ca, Mg free PBS, cut into even smaller pieces about (1mm) and after attachment add DMEM medium containing antibiotics, 10% FCS (I use 1 well of 24 wells plate). After about 4 days the cells start to appear around the explants and then I change the medium (then every 3 days) and after about 5 more days before they reach even 40% confluency the fibroblasts appear and start to overtake. So I use selective trypsinisation but in the process lose some keratinocytes as well. Unfortunately, after about 3 the fibroblasts re-appear.
How, can I get T-75 flask of pure keratinocytes starting with such small tissue size? What I am doing wrong?
I tried to swith to K-SFM after about first week (gradually and in one step) but without positive results ie. the growth of fibroblasts is still supported and keratinocytes do not multiply quicker.
I would appreciate any suggestions how to solve my problems.
joannanb on Apr 6 2010, 03:15 AM said:
When I receive the tissue I trypsinise it for 2 hr at 37C, then using forceps separate dermis, wash in Ca, Mg free PBS, cut into even smaller pieces about (1mm) and after attachment add DMEM medium containing antibiotics, 10% FCS (I use 1 well of 24 wells plate). After about 4 days the cells start to appear around the explants and then I change the medium (then every 3 days) and after about 5 more days before they reach even 40% confluency the fibroblasts appear and start to overtake. So I use selective trypsinisation but in the process lose some keratinocytes as well. Unfortunately, after about 3 the fibroblasts re-appear.
How, can I get T-75 flask of pure keratinocytes starting with such small tissue size? What I am doing wrong?
I tried to swith to K-SFM after about first week (gradually and in one step) but without positive results ie. the growth of fibroblasts is still supported and keratinocytes do not multiply quicker.
I would appreciate any suggestions how to solve my problems.
You could try live cell sorting to get a pure population, if the cells can survive the process. There are magnetic bead based systems, or you can just use FACS. Is there a cell surface marker specific for keratinocytes?
Explants are mad ol'skool, and not very efficient.
Try disassociating the cells in the epidermis using a trypsin enzyme, then pass through a 70 micron cell filter to get rid of the large sections of epi that are left.
Or if you are not harvesting from patients and just need cells you could just buy some, we use gibco HEKn and often get 25+ passages from them