Hello,
I have a ~2 g frozen pellet of CHO cells overexpressing a membrane protein (stored at -80°C) which will be used for membrane vesicles preparation. I need to resuspend the pellet, split it into 2 aliquots and then refreeze them in liquid nitrogen (I know it is not the best, but I have no alternative because I need to carry half of these cells in another facility for a different membrane preparation protocol). I´m not an expert on cells, so I´m trying to figure out what would be the best way to do this avoiding as much damage as possible to them. Should I resuspend the cells into a PBS-glycerol solution and then refreeze them like this or is it better to thaw them before refreezing? Or there are other - and better - options?
Thank you for your support,
Rocco Zerlotti
I would thaw the cells with gentle agitation until there is just a small amount of ice left floating .The mix is then at less than 0Centigrade. When the ice finally melts mix gently and briefly and remove the cells that you want to grow on., Then just refreeze the rest of the cells for later use. Slow freezing ( ideally 1c per minute ) can be done in an inflated polystyrene box with the sample surrounded by paper tissue in a -80 freezer overnight then transferred in to liquid N2. If your lab has a container of propanol surrounding dry tube holders then these also work well to produce slow cooling in a -80 freezer.
With the cells to be grown it is better to add small amounts of culture medium to the removed cells with gentle mixing so as to minimise osmotic shock until the total volume of the cells is at least doubled before adding to the culture medium in the TC flask
Hi Rocco,
It would be better to resuspend the CHO cells in medium Ham’s F12
Nutrient Mix medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and 10% DMSO. Or in a Complete ready-to-use cryopreservation medium as Recovery Cell Culture Freezing Medium (Gibco), based on a Dulbecco’s Modified Eagle Medium with optimized levels of fetal bovine serum, and 10% DMSO (10%).
Hello
You need to thaw the cell pellet before refreezing.
Thaw the CHO pellet at 37 degree celsius in the water bath. Add complete medium to the pellet consisting of HAM'S F-12 +10 %FBS + 1% penstrep. Tap the pellet gently. Centrifuge the cell suspension at 1500 rpm for 10 minutes. Discard the supernatant. Tap the pellet gently. Add the freezing medium meant for CHO cells. The freezing medium consists of Ham's F-12 + 10 % FBS + 1% penstrep + 10% DMSO. The freezing medium must be prepared before thawing the cell pellet and it must be kept on ice.
Add 2 ml freezing medium to the cell pellet after tapping gently. Divide the cell suspension in two freezing vials of 1.5 ml capacity.
The freezing vials need to be pre-chilled.
Freeze the vials containing the cell suspension gradually at 1 degree C per minute using Mr. Frosty container overnight at - 80 degree C and transfer the vials to liquid nitrogen the next day. If you do not have Mr. Frosty, you can place the freezing vials at - 20 degree celsius for 1-2 hours and then keep them at - 80 degree C overnight. Transfer the freezing vials the next day to liquid nitrogen. The transfer of the vials to liquid nitrogen has to be rapid without wasting much time.
I hope this is helpful.
Best.
Simple refreezing is not recommended, as it is likely to kill the cells. Also, the number of cells, even if they do survive, may not be large enough to restart a culture. Instead, grow the cells for 2 or 3 passages, then freeze the fresh cells by standard protocoll. This will also increase the number of vials of frozen cells, in case you need to restart them later.
Rocco Zerlotti
It is not good idea to refreeze the cells immediately after thawing. it is not recommended either. You may need to revive them first and propagate the cells . Then you might like to freeze the cells .
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