fresh foreskin, short decontamination procedure using povidone iodine and EtOh, overnight 4 C incubation in dispase II 1 ug/ml diluted in PBS w/ Ca, Mg for dermal-epidemal separation, epidermal sheet further digested with typsin/EDTA 0,05% 37 C 30 mnt properly inactivated with FBS ...culture perform with Epilife media with HKGS in collagen type I coated dish...resulting culture can only survive for 7 days before died out...opinion greatly appreciated
Hi Indra, are your keratinocytes adhering and not dividing? Are they dying through apoptosis?
Primary cells can be very particular about what they adhering to.
Keratinocytes also adhere to laminin
http://www.pnas.org/content/87/17/6888.full.pdf
Hi thank you, they are adhering and dividing very slowly...its a bit frustating actually
i have varied between collagen I and IV for coating..none help though
Hi Indra, its probably not due to the media, since companies selling keratinocyte media (Life Technologies and ATCC) basically have the same formulation.
This problem has to be occurring between the steps of digesting the foreskin to plating the cells
Things that I can think of:.
1) FBS was not heat inactivated. Heat inactivation of FBS destroys the complement. If the cells are dying without showing the usual apoptotic bodies, then complement might be the problem.
2) Contaminated cultures. Does the media turn color upon incubation? Are there foreign bodies in the culture? Yeast, fungi and mycoplasma are the most common contaminants in tissue culture.
A quick test for contamination is to take a sample of you cell supernatant and put in a sterile flask. Take a sample of your media and put in a sterile flask, If you cant see any evidence for contamination, look for apoptosis.
actualli i did use regular FBS to inactivate trypsin during epidermal digestion, okey than i'll try to inactivate next time.. thanks a lot
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