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Thawed cells spent 4 hours extra in DMSO.. problem?


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4 replies to this topic

#1 thebromar

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Posted 30 October 2012 - 11:10 AM

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

#2 madelingirly

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Posted 30 October 2012 - 02:39 PM

Dear
thebromar,

Once I got cells from ATCC, and the protocol was just like that, start culturing without removing of DMSO, and I did this and it was fine.
Then I searched online and I read that cells can withsatnd DMSO for 12-24 hrs, so I tried with another type of cells, I took my cells from liquid nitrogen, cultured them without removal of DMSO, just putting it in medium.
and my cells were fine too, my cells did not die.
I guess this is not a common step to do, but what I mean that sometimes there are types of cells very sensitive like those I got from ATCC and better to be culture without removal of DMSO for 24 hrs before u can remove ur DMSO

#3 bob1

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Posted 30 October 2012 - 02:56 PM

As Madelinegirly says your cells will probably be fine. However, this depends on the cell line and the concentration of the DMSO in the medium. As it was 1 ml of freezing mix in 30 ml, you are likely to have about 0.3% DMSO which should be fine for most lines

#4 thebromar

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Posted 30 October 2012 - 05:47 PM

Dear
thebromar,

Once I got cells from ATCC, and the protocol was just like that, start culturing without removing of DMSO, and I did this and it was fine.
Then I searched online and I read that cells can withsatnd DMSO for 12-24 hrs, so I tried with another type of cells, I took my cells from liquid nitrogen, cultured them without removal of DMSO, just putting it in medium.
and my cells were fine too, my cells did not die.
I guess this is not a common step to do, but what I mean that sometimes there are types of cells very sensitive like those I got from ATCC and better to be culture without removal of DMSO for 24 hrs before u can remove ur DMSO


Perfect. So actually, my cells were in DMSO for less than 24 hours, probably between 20-22. So, should be okay ,despite staying in 4h longer than I wanted. Thanks!

#5 leelee

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Posted 30 October 2012 - 06:20 PM

For what it's worth, I always just thaw my cells straight into media and don't even bother replacing it at all. I simply wait until my cells are ready to split (which can be up to 3 days) and then passage as normal.




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