While culturing immortalized ameloblast lineage cells from frozen stock (liquid nitrogen), despite there being good attachment numbers, subsequent proliferation of the cells is slow to negligible. These cells have a tendency to proliferate quickly when seeded with a moderate cell density. Previously, we were able to successfully culture these cells under identical conditions (low glucose DMEM enriched with 10% FBS and antibiotics). Currently however, the cells do not attach to plates unless high glucose media is used. And the attached cells are not proliferating despite a good proportion of cells with tight cell-cell contacts. Any suggestions on what other parameters to change are much appreciated! Thanks!
In my cell culture experience, I think supplementing with additional 10%FBS in the culture media helps if they come out from a frozen stock and has attachment issues.
How much time are you giving these cells for attachment?
Hi,
FBS has an important role in the attachment of cells. In case of attachment issue in the beginning, you can increase up to 20%, then back to normal once everything is fine. Do you use heat-inactivated FBS?
Apart from that, you had better check that your cell culture flask, petri etc. is tissue-culture treated.
Cells might be stressed. Even if they attached, they may not proliferate. You may need to give some time for recovery.
Cheers,
Aman Sharma Thanks for your suggestion! I give them overnight to attach and thus far we've been able to successfully culture from frozen stocks with just 10% FBS supplementation. I tried to increase FBS levels to 15%, and also use high glucose DMEM —although this does aid in better attachment compared to low glucose DMEM with 15% FBS, subsequent proliferation doesn't occur. The attached cells go on to not proliferate and end up detatch from the plate even after replacing the media.
Because of this, I switched the media to F12-K and now I'm able to successfully culture and subculture these cells.
Mert Burak Ozturk Thanks for your suggestion! I do use heat inactivated FBS and I did check to make sure the plate, incubator and other equipment used are in good condition. Switching the media to F12-K seems to work for the time being, I'll have to re-characterize the cells to make sure the media switch isn't impacting the expression patterns of these cells.
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