I am working on mouse embryonic stem cells (R1) and have been facing a lot of problems of late in its culture. After reviving, the cells grow fine on feeders for about 2passages. In the third passage, they start dying off, their morphology looks like apoptosized cells, colonies disintegrate and a lot of debris is formed in the media. I have checked for mycoplasma but the test results came negative.
I grow the cells in high glucose DMEM supplemented with 15%FBS, 1%NEAA, 0.1%Beta-mercaptoethanol, L-gulatmine and LIF. This behavior has been quite consistent and I have also tried with different batches of cells. Can anyone figure out what might be going wrong? Moreover, its only the ES cells are dying off; underlying MEF feeders seem fine. Ther's also no visible contamination.
Is there any strain of mycoplasma that is not detected by the conventional PCR test kits?
Yes, I actually meant 0.1mM beta-ME; the stock b-ME is 100mM and I dilute it 1000X.
I agree with Sunny Sun-Kin Chan, try to use a synthetic serum like KSR, and see what happens... If your cells remain like this, try another mycoplasma test... There are lots of mycoplasma strains and sometimes the level of contamination is really low and can not be detected but still infect your cells..
In anycase try to renew all your reagent of media and see if anything gets better.
Best of luck. Keep us updated of any changes .
I bet it is something wrong with media. I would suggest to check formulation and concentration of additives if you are preparing media from the scratch. But most probably issue is in a quality of FBS.
Best wishes,
Lena
please check your incubation condition such as incubator or osmosis etc.
You can try another stem cell line, I work with ES-D3 and they grow well on feeders using either FBS or SR. Also, try to use early passage cells from your stocks and see if this makes any difference
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