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..
I added the wrong concentration of DMSO in my cell storing step (1% DMSO), it will harm my cells, right??
How can I add more DMSO inside the cryo-tubes? Or I need to start a new cell line storage?
Universitas Islam Negeri Maulana Malik Ibrahim Malang
U.S. Food and Drug Administration
Leiden University Medical Centre
Sanko University, Gaziantep, Turkey
University of California, Los Angeles
Kris Cahyo Mulyatno
Medical College of Wisconsin
The University of Edinburgh
Fahad A. Alhumaydhi
University of Surrey