Cell debris here, there, everywhere!? - (Jun/28/2011 )

I use a stably transfected CHO cell line and trying to thaw these cells from liquid N2. I follow all the protocol - quick thawing at 37C, centrifugation for removal of DMSO - but the next day I see cell debris like everywhere. It fills the entire plate and the cells are rounded up and definitely appear to be undergoing apoptosis. I know freezing probably didn't make them happy but I followed the proper protocol there as well - media with 20% FBS and 10% DMSO with freezing at 1C per min. using an appropriate freezer container at -80C and dumped them in liquid N2 following day.

Biggest worry is i have used up all the cell stocks we got from ATCC and all the stocks i made from them seem to disintegrate forming cell debris every time i thaw an ampule. These are precious cells - stably transfected - we can't get them back unless we do cloning again. Stumped !!!!?!?!?!?!


Additional Details
I normally used T-75 for thawed cells with 12ml media. We ran out of T-75s so i now use 10cm petri dishes with 8ml media. I have also tried T-150 with 20ml media. I see cell debris in all 3 cases

I don't think its contamination as i don't see any bacteria or the media doesn't change color or anything until and unless all cells die and lift off the plate. No one else has a similar problem although we share FBS and P/S, the hood, incubator etc. I'd really doubt it was mycoplasma coz my cells don't even last 12 hrs before dying out completely. It seems to be a classic case of them not being happy fr some reason.

When I have a cell line that doesn't thaw well, I do the following:
I thaw the vial in a 37'C water bath, I do not swirl the vial I just hold it steady. I then transfer the contents of the cryovial to a 50ml tube. I drip wise add 4mls of cool or room temp media. I take a sample to check count and viability. I seed the cells into the appropriate size flask (adjusting the volume for the flask). The following day, after checking to see how the cells are doing under the microscope, I remove the media that contains the traces of DMSO and replace it with fresh warm media.

I don't thaw the vial in a water bath, just hold it in my hand, flick open the vial and then drop media from a sterile pasteur. Fill the vial (we fill 1ml of a 2ml cryovial) and move to a bigger volume. Then put everything into a T75.

I change media the next day to remove DMSO.