N2 storage of murine cell-lines

Basavaraju Shankarappa bsh at MED.PITT.EDU
Thu Aug 27 08:00:56 EST 1992


> >I've had problems in successfully freezing macrophage cell cultures so they 
> >can be successfully resuscitated. The problem does seem to lie in the actual freezing. The cells are harvested in the log phase, suspended in a glycerol freezing mixture at five-fold concentration then left overnight (well insulated) in a -70C freeze
> >Can anybody suggest alternative procedures? The alternatives facing me at present are constructing a "freezing box" which will cool at the correct rate in a -70C freezer (feasible) or persuading the department to buy a commercial cell freezer (less f
> >
> >Sean Shaw
> >Microbiology, Reading University
> >skrshaw at sksscsc1.rdg.ac.uk
> Forget the need for commercial freezing machines.
> For many years I have been able to successfully freeze a wide range of
> mammalian cells including mouse and cattle cells, monoclonals etc. with
> excellent retrieval rates (sometimes > than 90%). The cells are simply
> suspended in sterile 90% Foetal Calf serum, 10% DMSO, frozen overnight at
> -70*C in a styrofoam container and then stored in liquid nitrogen (some
> have been down more than 10 years and still revive).  Retrieval is by quick
> thawing in a 37*C water bath until JUST thawed (do not warm to 37) and
> IMMEDIATE resuspension in fresh COLD medium (DO NOT pellet the cells before
> resuspension in the fresh medium).  If the dilution is more than 10 fold
> i.e. the DMSO is reduced to below 1% you do not even need to spin down and
> wash the cells, just plate directly.  If not pellet gently, resuspend
> gently and then plate as per normal.
> 
> I hope that this works for you as it does for me.
> 
> Cheers, Klaus.
> ----------------------------------------------------------------------------
> Klaus Matthaei
> Gene Targeting
> The John Curtin School of Medical Research
> The Australian National University
> E-mail: Klaus.Matthaei at anu.edu.au
> 
> "Don't try and fix it if it ain't broke".
I second Klaus' opinion with great pleasure.  I had actually forgotten
that I had used almost the same procedure which was given to me by
someone I cant remember.  But one thig is for sure.  I had very good results
with this procedure.  In fact we had a LN2 mishap and I was able to 
resuscitate one of the very important hybridoma cell line that was 
frozen with this procedure.  
	I also agree with Klaus' observation that you do not need to
spin the cells to remove DMSO.  I have been given to understand that
it just evaporates in the incubator, if sufficiently diluted.  I 
think it is more of a trauma for the cells to be spun rather than
diluting out the DMSO.  
	One difference from the above protocol I had followed was, after
thawing the vial (approx 1 ml), I added the contents directly to the 
flask that contained about 15 ml of the media.  

So here is another vote for BFS-DMSO
Raj Shankarappa
Univ of Pittsburg
bsh at phobos.med.pitt.edu

flask that conata



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