What's the 'best practice' when it comes to thawing frozen cells? What I usually is do thaw them one afternoon and split them the next day. Should I instead leave them in culture for longer before first change of media?
Also, how are you supposed to wash when thawing? I spin only once, but was told that I should at least three times, to make sure every trace of DMSO is gone.
1 time spin would be sufficient. But if the cells been frozen in liquid nitrogen, say few months, cell viability will decline after long storage and will take few days to really attached. I normally just add fresh media when washing (at least after 2 days thawing out). And you should leave the cells to attached as much as possible and not introduce stress by splitting them the next day.
Hi Isabel one spin is more than sufficient - indeed for may cells you don't even need to spin (if you freeze in 10% DMSO and transfer 1 ml of cells to i.e. 20 ml of media the final % of DMSO is 0.5%.. You could always remove this media (after the cells have adhered) and replace with fresh media to get rid of this.
I always leave cells a few days after recovery - you should not need to split them the next day if you are reviving them into the correct sized flask (i.e. 5x10e6 into a T175 not a T25 for example).
For some cells (non-adherent cells especially) I find that adding the revived cells to pre-buffered media helps their recovery a lot so it would not harm to do this for your cells.
By pre-buffered I mean 15-30 minutes before you get the cells from LN2 put the media you will use into your CO2 incubator to allow the media reach the correct pH/CO2 saturation (i.e. put 25 ml of media in in a T175 flask in the incubator or media in a 50 ml tube with the top loose).
Hope this helps,
Gary
One spin is enough if done properly. Depending on the type of cell (adherent or non adherent) I wait for the cells to attach to the flask, if they don't attach it could be a sign of trouble for adherent cell. Usually the cells start to attach already after one day, but if this does not happen I leave them 2 or 3 days. If they then still did not attach I try to replace the medium and see if they will attach then... if not then I am afraid the cells did not survive in sufficient numbers. Sometimes even the low amount of surviving cells will be able to make the cell line recover, but this requires a bit of patience.
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