Protocol Online logo
Top : Forum Archives: : Cell Biology

Freezing and thawing - Good thawing V% but...... (Nov/09/2006 )

Hello all,

I banked certain suspension cells. When I thawed a vial for testing, I got good # of live cells and good V% - when I counted a sample immediately after thawing. I thought the banking process was good. But the next day, when I re-counted a small sample (for post thawing - 24hr data), I only got 78% V% and cells did not seem to multiply - infact they were lesser than expected - as some cells died - shown by decreased V%. And no, I did not spin down to remove the DMSO because after thawing I put the whole frozen cells into 20ml of media - isn't that dilution enough? I don't understand. Any suggestions? The DMSO in frozen medium was only 5%.

Thanks

-scifi-

QUOTE (scifi @ Nov 9 2006, 09:31 PM)
Hello all,

I banked certain suspension cells. When I thawed a vial for testing, I got good # of live cells and good V% - when I counted a sample immediately after thawing. I thought the banking process was good. But the next day, when I re-counted a small sample (for post thawing - 24hr data), I only got 78% V% and cells did not seem to multiply - infact they were lesser than expected - as some cells died - shown by decreased V%. And no, I did not spin down to remove the DMSO because after thawing I put the whole frozen cells into 20ml of media - isn't that dilution enough? I don't understand. Any suggestions? The DMSO in frozen medium was only 5%.

Thanks


I think you'd better remove DMSO by centrifugalization ,for DMSO is harmful to cells.Otherwise you can make a contrast between the two methods,and count the ratio of living cells.
Good luck!

-wing111-

I don't remove DMSO either but I change the medium the day after. It is normal that your cells grow slowly after thawing: you should give them a couple of days to recover. Some people use, to freeze cells, 5% DMSO in culture medium (DMEM+10%FCS); you could try, as I do, 95% FCS + 5% DMSO

-dnafactory-

I centrifuge to get rid of DMSO and I also see in a delay in the growth of the cells.
They need time to recover

-Missele-

QUOTE (scifi @ Nov 10 2006, 05:31 AM)
Hello all,

I banked certain suspension cells. When I thawed a vial for testing, I got good # of live cells and good V% - when I counted a sample immediately after thawing. I thought the banking process was good. But the next day, when I re-counted a small sample (for post thawing - 24hr data), I only got 78% V% and cells did not seem to multiply - infact they were lesser than expected - as some cells died - shown by decreased V%. And no, I did not spin down to remove the DMSO because after thawing I put the whole frozen cells into 20ml of media - isn't that dilution enough? I don't understand. Any suggestions? The DMSO in frozen medium was only 5%.

Thanks


what is the dilution factor of thawed cells in 5% DMSO (volume?) to 20 ml medium? To be save, DMSO should not be > 0.1 % after dilution

-The Bearer-