Attention! Guy wanting Cryopreservation help!

IVAN at MNI.lan.mcgill.ca IVAN at MNI.lan.mcgill.ca
Wed Sep 1 20:24:13 EST 1993


Attention FGARBREC (sorry, I dumped your original post and can't
remember your name)!  Your reply address doesn't work.

My original message, which has been reflected back by your mailer 3
times now, was:

> I have, over the past couple of years, gotten a number of murine
> hybridomas to grow well in various serum free media, but have been
> unsuccessful in cryopreserving serum-free adapted hybrid cell lines.
> I use the standard 10%DMSO/90%FCS as a cryopreservative,

This is really strange.  When we were generating MAb hybridomas
(mouse-rat fusion; really tough), we could both adapt what few
hybrids we got to serum-free and were able to successfully freeze and
thaw whenever we felt like.

Our freeze-down method is slightly different.  There are two
cryopreservatives:

    Freezing Media              Cell Media
    10% DMSO                    80% FCS
    70% FCS                     20% DMEM or RPMI 1640
    20% DMEM or RPMI 1640

Everything is filtered through Nalgene 0.22 um nylon units.  Freezing
Media and pipets for it are put into -20oC freezer 30 min before use.

Cells are harvested as usual and resuspended in Cell Media at approx.
2 to 3 million/mL.  0.5 mL are then loaded into each CryoNunc vial (1
million/tube).  0.5 mL Freezing Media is then dribbled down the sides
of each vial (if you can make a layered drink or load Ficoll
gradients from the top, you can do this).

CryoNuncs are kept at -70oC overnight and then transferred to liquid
nitrogen.  We used to use a vapor system but switched to a liquid
suspension system because it was cheaper.

If you have any problems or comments, please call or write; my phone
number is on the signature.  Have fun.


- ivan

------------------------------------------
Ivan Shaw "The Angel of Death"
Neuroimmunology Unit
Montreal Neurological Institute
Montreal, Canada  H3A 2B4
------------------------------------------
Internet e-mail:    Ivan at MNI.LAN.McGill.CA
Telephone:          (514) 398-3002
FAX                 (514) 398-7371



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