The idea that DMSO will be less effective at driving water out of the cells during freezing, therefore DMSO should be fresh is reasonable, but I must say, I have been using the same bottle of DMSO since 1997 with no problem whatsoever with my cell lines. We always freeze in a final concentration of 10% DMSO/40% FBS/50%media containing cells. We always prepare the DMSO/FBS fresh on ice and freeze either with one of those alcohol freezing containers or with styrofoam containers with equal success.
I have always aliquotted cells in 1 ml aliquots, and when thawing, I don't bother to spin them down to remove the DMSO, but rather simply change the media the next morning. Works quite well for HIT-T15, INS-1, MIN6, beta-HC alpha-TC1-6 and 1-9, Beta-TC6.
@Chopie Hassan - I routinely freeze lung fibroblasts in 10% DMSO, 40% FCS, 50% Full DMEM media (which itself contains 10%FCS). And the cells are perfectly viable once thawed. (not sure about your specific makeup of freeze media, but this works)@Fahran Haider - No. Cells, once in the freeze media, should be transferred as quickly as possible to the -80C in a suitable freezing container that cools at around -1C/min. Then, once frozen, should be transferred to liquid nitrogen for long term storage. (Something like this Mr Frosty in the link provided..)
@Farhan I must confess I have never even tried to store at -20C so can't answer that one. I was always advised not to!
@Farhan based on my experience, you can store cells in -20 maximum overnight, then viability drops down...Some protocols suggest that you cool cell @+4 for 1h, then trasfer to -20 for 3-4 hours (max) and then -80...In any case, I agree with Mattew..better not to do..
Universitas Islam Negeri Maulana Malik Ibrahim Malang
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