Thawed cells spent 4 hours extra in DMSO.. problem? - (Oct/30/2012 )

Hi there,

Is it an issue if cells are left for a few extra hours in DMSO after thawing? I'll give a rundown of the protocol

1ml cells w/ dmso thawed + 30ml cell culture media -> Overnight incubation -> First thing in the morning (9-10AM) media should be changed to fresh media to remove DMSO

At the concentration it was present overnight, are all cells likely to die or are there any major consequences to leaving the cells in DMSO media for four extra hours, until 1-2PM?

I know I could wait and see but I'm looking to put my mind to rest

Thanks

I would say it depends on what concentration of DMSO you have in the frozen stock. We normally freeze our cells with 10% DMSO added to DMEM+FBS (although I've heard fully concentrated FBS works better), then thaw 1 ml with 9 ml fresh media for a 10 cm dish. We'll regularly leave that overnight. So that's roughly 1% DMSO.

If you're diluting a stock at 10% DMSO into 30 ml, your final DMSO concentration will only be 0.3%, much less than what we leave them in. I'm sure they'll be fine, although different cell lines will have differing sensitivity to DMSO.