Donald Baumann dbaumann at mail2.sas.upenn.edu
Wed Apr 15 14:29:19 EST 1998

Kalpit might have the right idea if your freezing mix is the problem, 
which I don't think it is.  Your freezing procedure is almost exactly 
what I use. The problem is probably in the cells themselves or in your 
thawing procedure.  If your worried about losing some of your cell line 
my advice is to avoid all the spinning and rinsings after thawing; just 
thaw and put directly into culture, just make sure to add a little extra 
media to further dilute the DMSO.  I often do this when I have the same 
problem as you and it works.

Kalpit A. Vora (K_Vora at lac.jci.tju.edu) wrote:
: In article <Pine.GSO.3.96.980413132953.29697B-100000 at dilbert.ucdavis.edu>,
: "L.A. Pike-Nobile" <ez063669 at mailbox.ucdavis.edu> wrote:

: > I am getting extremely variable rates of viability from my B cell lines
: > when I thaw them after storage in liquid N2. I freeze them in 50% media/
: > 30% FCS/20% DMSO. I freeze them gradually (I use on of those little
: > ethanol-jacked freezing things that claims to give 1oC/hour) and I thaw
: > very rapidly (37 water bath) and rinse them thoroughly. 
: > 
: > AND YET--I still get some vials with less than 1% viability while others
: > are fine. Some lines are better than others, but I see this problem even
: > within the same line, frozen at the same time. I'm worried about
: > completely losing some of my more fragile lines.
: > 
: > Are there any tricks I should know about?
: > 
: > Thanks,
: > Larry
: Dear Larry,
: We routinely freeze our hybridoma in fetal calf serum containing 5%DMSO
: and nothing else. Also We pellet the cells resuspend in the above freezing
: medium and leave it overnigt at -70 before transfering to liquid nitrogen.
: It can remain at -70 for 3-4days without effecting the recovery. Also try
: and freeze the cells when they are young and growing in exponential phase.
: I donot see any problem in the way you thaw them out.
: Hope this helps.
: Kalpit

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