HUVEC Cell freeze & thaw

[Susan Hubchak]

In article <drnamba-1212961959510001 at anes16.kuhp.kyoto-u.ac.jp>,
drnamba at kuhp.kyoto-u.ac.jp (Tsunehisa Namba) wrote:

>    I culture HUVEC cells (Human Umbilical Endothelial Cell primary
> culture) in M199 media containing 20%FCS, 15microg/ml ECGS, 90microg/ml
> heparin and Pen Strep on gelatin coated plates.  I heard these cells can
> be stored frozen until use.  However after freezing these cells in 10%
> DMSO in culture media, non of the cells look viable.
>    Any suggestions?
> Tsunehisa Namba
> 
> -- 
> ***************************************************
> Kiichi Hirota, MD
> Department of Anesthesia, Kyoto University Hospital
> Sakyo-ku, Kyoto 606-01, Japan
> Phone: +81(Jpn)-75-751-3436   Fax: +81(Jpn)-75-752-3259
> E-mail: khirota at virus.kyoto-u.ac.jp

In our lab, we use a product from Biofluids Inc [1-800-972-5200 fax 
301-424-3619] called Cell Freezing Medium B cat# 133.  Essentially,
it's L-15 medium containing 20mM HEPES, 15% DMSO, and 2% PVP.  We
centrifuge the cells at high density (4-8 million/vial), resuspend the
pellet in half the final volume in growth medium and make up the remaining
volume with Cell Freezing Medium.  The vials are frozen overnight at -80 C
and then transferred to liquid nitrogen vapor for long term storage.
Hope this helps.

Susan