thawing & plating cell lines

Lesley Weston lesley at UNIXG.UBC.CA
Fri Jun 21 19:20:12 EST 1996

Another possible answer is to use 10% gycerine instead of DMSO in your 
freezing mix. Then you don't need to spin the cells to remove it, 
provided it's diluted in fresh medium when you plate. The speed of 
thawing seems to be at least as important as the speed of freezing; I put 
the vial straight from the liquid nitrogen into a beaker of water at 
about 37 C, and then pipette the cells into warm medium as soon as they 
are thawed. Hope this helps.

On Fri, 21 Jun 1996, Andreas Jordan wrote:

> In most cases we observed those effects on any mammalian cells, there 
> were some uncertainities during freezing procedure. For example often 
> a slight increase of the temperature during freezing procedure above 4 
> degr.C cause damage to cells by DMSO. Furthermore, the freezing speed 
> from 4 degr. C to -80 degr. C to liquid nitrogen is critical. We 
> always envelop the cryo vial boxes with thick cotton or paper towels 
> at the time interval between 4 and -80 degr. C to achieve at least a 
> temperature decrease of 1 degr C per minute. Faster freezing will also 
> damage the cells physiologically so that they come out dividing later 
> as usual. My last concern is speed of centrifugation after thawing 
> procedure. You should try to use the lowest speed possible for 
> sedimentation and avoid pipetting the cell suspension more than 
> absolutely necessary in order to minimze membrane damage of the 
> freshly thawed (mostly sensible) cells. Use 25 or better 12.5qcm 
> flasks (Falcon) for first passage to obtain best cell density, i.e. 
> cell growth.
> cram at PIPELINE.COM wrote:
> > 
> > 
> > Hello,
> >     my team and I are having trouble producing healthy-looking cell
> > cultures at plating time.  We thaw quickly, spin to remove DMSO and then
> > plate.  After one night, the cells are dividing slowly, and have a strange,
> > small, sort of compact morphology.  Overall, they are half the size as they
> > appear when healthy.  Only after another day or so do they have the nice
> > flattened look to them, where you can see the cytoplasm and their nuclei
> > are large, etc.  Then they grow to confluency, no problem.
> >     Could this be due to a low density at plating, perhaps too low?  Or is
> > it more likely caused by our cryopreservation?
> > 
> >     Any input would be appreciated.
> >     Thank you in advance.
> > 
> > Marc Rothman
> > <cram at>
> > Technician
> > Neurosurgery Research Labs
> > Dept. of Neurosurgery
> > NYU Medical Center
> > 550 First Ave.
> > NY, NY  10016
> >

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