help with cryopreservation - (Feb/12/2002 )
I have been having trouble with viability of my BRIN-BD11 rat beta-cells recently, and would like to know if anyone has a good method for freezing down cells that doesn't involve paying £50 for a Mr Freeze cryo tub [any budding home improvers out there who can suggest a way to build one?]
I have had very good success in freezing cells by simply putting the Nunc vials (or whatever you're using) into a
stryofoam box in a -80 C freezer for 24 hours then transferring directly to liquid nitrogen storage. The box should already be in the freezer before you start. I've done this a couple of hundred times and always had viabilities of at least 70%.
Agree with the last post. Even simpler, we used to place the tubes of cells over a canister of liquid nitrogen for an hour or so first, then to -80 for 5 hours and then to the storage canister.
i agree with nevermore. used that method tenths of times.
The technique mentioned really works well also take care that you have 950 of your cell suspension and 50 of sterile DMSO
i frz cells regularly by putting them in cryovials(at 4 deg C), transfer to-20 deg C(1hr),shift to -80 deg C for few days & transfer to liq nitrogen
The trick is not to freeze your cells too quickly. By putting them in a styrofoam box in the -80 freezer overnight, it allows your cells to freeze down slowly. Works great!! Liquid nitrogen next and you're in business!!
I agree with preethi, i find freezing the cells at 4 for few hours, then at -20 for overnight then at -70 for week or so then transfetring to liquid nitrogen results in better stability of cell membrane, by following this method one would prevent the cells from undergoing cold shock which can result in necrosis of cells.
My method is more simple. I directly put the tube into -80oC. Then keep there for years. The viability of the cells usually are more than 80%. I used this method for at least five kinds of different mammalian cells, it works very well. I have to mentioned that the medium what I used to store the cell were 90% of FBS and 10% of DMSO. Good luck.
Your post has partially clear my doubts on preserving cell lines in -80oC. My question is: Is this technique applicable to the MCF-7 cell line?. Pls kindly reply thru post or email (firstname.lastname@example.org). Thanking you.