Problem in mouse embryonic stem cell culture - (Dec/23/2014 )

Hello everybody
Iam culturing R1 strain of mouse ES cells. The problem iam facing is that the cells die after 2 passages. The culture conditions are the same as given in the protocol- 15%FBS, 1x beta mercapto ethanol(final concentration), 1x NEAA(final concentration) 1x Lif(final concentration), and the volume was made up with DMEM(lonza). For the feeders the MEFs were treated with mitomycin-c for 2 hrs. Washed thrice with 1x PBS and trypsinized for 30 sec and plated 20,000 cells/8cmsq.
Can you help me out with the problem.
Regards,
Dhanur. P. Iyer

Hi, did you check for mycoplasma contamination ?

Yes I did check for mycoplasma contamination but the result came negative.

Can someone please help me out

There are other options for culture techniques. See these publications. And this technology. Hope this helps! 

Leaders in 3D Cell Culture,  Synthecon