Maybe a problem with the culture buffer

Did check the presence or not of trypsin in cell culture medium?

We inactivate the trysin with 10% serum in dmem at equivalent volume, however we only centrifuged the cell suspension once..the pellet were resuspend in the culture medium and goes directly for seeding...

I am relying on dilution and washing to neutralize dispase and serum tor trypsin...

We had once - many years ago - a similar, serious problem with KCs from breast skin, which we cultivated serum-free in KGM.. Eventually , going through KGM-constituents, we found out that the producer had dramatically reduced the growth factor concns. (without notification) Restitution of former concns, made cells grow well again.

Nevertheless, we always checked for mycoplasms contamination (for some time, we also used a relatively specific antibiotic against mycoplasms: tiamulin).

good luck!

Thank you Inge & the others, weve form some modification on our protocol based on your comments..were gonna have another go this thursday..further suggestion are welcomed

I haven't work with keratinocyte culture for the past year, mostly with stem cells and blood cells, the common ground is that they are primary cells and not immortalized cell line.

I've several possible scenarios in mind that you may want to consider:

1) culture condition, beside contamination, could there be a simple human error or changes that occured during the cultivation, i.e mixing up of basal medium, percentage of serum, expired date of batch of medium and serum and additional amino acids or growth factors?

2) what about confluency and split factor? some cells like to be in contact with others, if you split them too early, while the confluency is still low or suboptimal, this will also cause the cells to lose their survival signal that ignites from contact to other cells and they will undergo apoptosis or cell death

3) how about the nature (cell cycle or turn over rate) of keratinocyte in vitro, since they are primary cells, how many passages can they endure before the telomerase (biological clock) inside the cells ran out and shortening of the telomere occured and the keratinocyte undergoes apoptosis?

4) is there any known toxic condition in the keratinocyte culture environment? for example, when we cultivate hematopetic stem/progenitor cells and differentiate them, some of the cells did not like the condition, and were necrotic, release their content and toxic for other cells or at least gave a suicidal signal to other cells in our culture flask.

I use 0.1% trpsin in PBS to digest trimmed foreskin 24 hrs, then separate epidermis from skin and isolate keratinocyte by di-associating epidermis, and finally inactivate trypsin with 10%FCS. It works very well for me nearly 10 years. never used dispase.

Sometimes amphotericin B is a bit rough on the cells, so we don't use it unless we really need to. It's never a permanent additive.

@Meilang Xue: what do you mean by di-associating epidermis? mechanical or enzymatic?

@Daniel & Mba Nana: thank you

Hello

There are two possibilities

1. I think while isolating cells, a lot of cell debris was seeded with the cells. These cells are very difficult to handle as they are VERY VERY Sensitive. I would suggest decrease your isolation time (25% reduction). I would also suggest a media change after day 1-2 (Depending on attachment).

2. I would recommend use of Trypsin Inhibitor. Works way better than FBS. 

Hello Indra

Has the problem solved yet? If so, can you share the experience? I encountered similar problem recently, with the Epilife medium.

Hi Hao Tan, Thanks for reaching out

The problem is solved. However we cant pinpoint the culprit definitely.

First of all, if you used Epilife medium than i assume that used also HKGS. Which i realized at that time that they change insulin to IGF.

However addition of Insulin (from ITS) doesnt fix the problem.

Secondly is trypsinization, we reduce concentration from 0.25% to 0.05% as working concentration. This seem improved condition, we currently used 0.05% as a rule. Gibco seem agree with this because recent product also used this concentration.

Third is we use a rule in our lab that Isolation can only performed at monday and tuesday to avoid friday as DIV1 (we used two step isolation that involved overnight dispase in rerigerator). Somehow we never encountered the problem again

All in all, primary KCs need a lot of attention, but we love them and healthy KCs give if beautifull sight under the microscope :)

Hope this help you. Good luck

@ Indra Kusuma , thank you for the update. Good to know you have fixed the problem.

I did used HKGS with Epilife, actually the medium is the same with yours except the L-Glutamin , could that be the problem? Do you still use the same medium, with insulin added?

For trypsinization, I used the TrypLE, which is much gentler than trypsin/EDTA, but that seems not helping.

The last thing is I checked the temperature inside the incubator for a while, which turns out to be around 35 ℃. Maybe primary keratinocytes are very sensitive to temperature or mycoplasm contamination, perchance?

Hao Tan,

I find that L-Glutamin indeed necessary, but not insulin, which we dont use anymore

I believe temperature also very important, because we always make sure that medium is adequately warmed and equilibriated before use

Mycoplasma indeed a problem, but in our experience the real problem usually closer, you should recheck all that are in contact to the cells are in good condition

@ Indra Kusuma Thanks! By the way, in your experience, how long the Epilife medium can stand good in 4℃ refrigerator, with HKGS and other stuff added?

Hao Tan, I think it is about a month