Hi,
Am working with primary normal human epithelial keratinocyte (NHEK) and I’m using KBM-Gold as cell culture medium but I'm having problems with cell proliferation. They stop proliferating after passage 4/5. Can anyone advise me which medium to choose to have more passages?
Thank you for your answers
They have a finite lifespan, I've cultured primary foreskin keratinocytes which we isolated ourselves and have cultured commercial HEKn, in my experience the commercial ones stopped proliferating quite early, also around 4/5 passages. The ones we isolated ourselves grew up to 8 usually. I used Gibco Keratinocyte-SFM medium (17005-075). What you could try is adding the Rock inhibitor Y27632. There is a paper on using this inhibitor to increase the lifespan of primary keratinocytes without transforming them into cancer cells. Never tried it with keratinocytes, but used it for HAE culture. I have the paper somewhere, let me know if you can't find it.
They have a finite lifespan, I've cultured primary foreskin keratinocytes which we isolated ourselves and have cultured commercial HEKn, in my experience the commercial ones stopped proliferating quite early, also around 4/5 passages. The ones we isolated ourselves grew up to 8 usually. I used Gibco Keratinocyte-SFM medium (17005-075). What you could try is adding the Rock inhibitor Y27632. There is a paper on using this inhibitor to increase the lifespan of primary keratinocytes without transforming them into cancer cells. Never tried it with keratinocytes, but used it for HAE culture. I have the paper somewhere, let me know if you can't find it.
Y27632 will extend the keratinocyte life span, but we found that it altered the signal transduction pathway activity in the cells, and was not reversed when removing the drug. I would carefully consider what you want to use the cells for before using a Rock inhibitor.
ah really? like i said, never used it with keratinocytes, but find it used in a lot more cultures other than keratinocytes (working with stem cells now and even there it is used after thawing cells). just out of curiosity, did you publish your findings? would like to read it!
Thank you for your ansewrs,
I prefer not to use any drug. I don't want anything to interfer with my research analysis.
I was wandering if we I could extend the Keratinocytes lifspan by changing a cell culture medium.
Just a little comment. If you say you are working with "primary" foreskin keratinocytes than it really does not make sense to talk about passages. These cells are only primary when in P0 ;-)
Are you saying primary because you extract them yourself? Because, if you are buying them, they can often arrive with lots of population doublings in them already, which will affect the number of passages you can do.
I'am byuing a NHEK (Frozen).
And exactely PDs affect the number of passages.
Hi Zineb, P7 is (lower) average for NHEK , that depends on your population doublings though, and there is large donor variation, even for neonatals. But for media, you might try either DermaLife K Medium from Cell Systems or KGM from lonza, we made OK experiences with both.
Hi Florian, Thank you for your answer. Actually, from medium, I'm using KGM from Lonza but my NHEK stop proliferating or proliferate slowly at the passage 5. and I'm respecting the recommend initial density (3500 Cells/cm2). I just tough of change to Epilife medium but I'm hesititing. Can you please tell me more about your one experience with NHEK culture. I'am used to work with HaCaT. thank you again.
Keratinocytes will grow well on 3T3 feeder layer, but we want to try xenofree material, that's why we used Epilife-S7.
We have used Epilife and S7 for human keratinocytes (we isolated ourselves) without feeder layer, but failed. Then We used Epilife - HKGS (contains xeno material) also without feeder layer, the result was variable, one time good, another time very bad, Epilife has batch to batch variation, if you are lucky, you get a good batch, and if you are not you get a bad one.
For Epilife, the plastic ware should be coated.
Grow your normal primary Human epithelial keratinocyte with rheinwald & Green method (with 3T3 feeder plus DMEM+F12 with supplements). it will give you a better result than any other feeder less system. so try for it and you will get success.
In my hands, KGM is not the best. F12 looks better but no guarantee. We make our own media which is so called E-media. This media works very well when we co-culture HFKs with J2 feeders. I can get up to 13 passages.
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