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.
Universitas Islam Negeri Maulana Malik Ibrahim Malang
University of Florida
Center for Research and Advanced Studies of the National Polytechnic Institute
Sanko University, Gaziantep, Turkey
Fahad A. Alhumaydhi
University of Surrey
Medical College of Wisconsin
University of California, Los Angeles
Kris Cahyo Mulyatno
Wayne State University