Problem with thawing cells from liquid nitrogen? - (Mar/18/2008 )
I have been thawing two cell lines (MCF-7 and HeLa) from liquid nitrogen, but I don't have much live cells. almost all the cells have died.
I usually take the vials out of the liquid nitrogen and put in water bath immediately. when the ice is melting and the DMSO is still not harming the cells I add the media + FBS and centrifuge(5min,1000RPM). then I resuspend and again centrifuge for another 5 min at 1000 RPM. then add media+10% FBS+ 1% antibiotics.
but even when I change the medium after 12 to 24 hours the number of cells that are attached to the flask is very limited.
what can be the problem?
Try not to spin the cells after thawing because spinning may do more harm than just leave the DMSO in the medium. Another cause may be that your cells were not properly frozen.
yes, thats what I thought too
I can't speak for the HeLa, but I work with MCF-7 and have to agree with pcrman - don't bother spinning. Once the vial is completely thawed, add it to your flask. Let the cells adhere, and then feed the cells the next day to remove the media with DMSO.
I wouldn't bother with the spinning either. Just put extra media in the flask to dilute out the DMSO. I use 20mL in a T75 when thawing cells. Then change the media the next day.
Otherwise it could be a bad freezing.
MCF-7 cells may need insulin to grow happily. Helas are like weeds and could probably grow in water
On my hands, I thaw HeLa successfully by spinning, nevertheless I spin only once.
Usually I completely thaw the vial in water bath, dilute the content in cool medium, then spin down, usually with shorter time and lower speed than routine passaging (but it should be fine even with the same settings, I tried before). Discard the medium, Resuspend the cells and grow.
I don't want to scare you, but usually for the failure of thawing, it is the problem of how the freezing process of those vials are performed but not the thawing process.
YES, PROBABLY THE PROBLEM WAS WITH THE FREEZING,
HELA CELLS SURVIVED BUT ALL MCF7 CELLS DIED. HOWEVER SO MANY OF THE HELA CELLS HAD DIED TOO, BUT I MANAGED TO GROW THE REST OF THEM. NOW THE FLASK IS CLNFLUENT.