Cryoconservation - Problem - (Aug/22/2007 )

Hi,

I have chicken B cells. Due to technical reasons, I have had to freeze all my cells (with DMSO). After 5 weeks, I started to defrost my cells. It is high likely that my -80°C Freezer was defect for an unknown period of time. Nevertheless, I checked the vitality of my defrosted cells with Trypan blue and I have for example a vitality of 70 - 90 %. After one day, the vitality was observed to be strongly decreased (to only 10%). Even after one week, no cell growth could be identified.

Does anyone know a procedure as to how to rescue my frozen cells?

Thanks

Juliano

Nope if they're dead they're dead.

Your trypan staining right after defrosting is meaningless. The one after 1d gives you a real idea. You can try to recover them in 20-50%FBS, but I wouldn't use them even if you can recover some cells from that. Who knows what you selected for during such extended cell stress?

Best bet is to find new ones from somewhere - and establish some backup systems for your freezer (btw cells should not be stored long-term in a -80 freezer but in liquid Nitrogen).

you should thaw your cells fast. half immerse your vial in 37C waterbath for not more then 5 min. resuspend in pre-warmed medium as soon as the vial start to thaw/half thawed...

How many cells do you defrost and put in one flask/dish?
I've noticed that, after defrosting, cells prefer to stay "close to each other"... or at least that's what they told me
ok, stop with jokes, I suggest you to seed them at higher cell density than the one you set for usual transfer.