Freezing and Thawing cancer cells

 I have been freezing  cells  in a freezing medium consisting 10% DMSO, 20% FBS and 70% RPMI or DMEM.  The cells will be stored overnight at -80^oC and then transfered to liquid N₂ .   We make our own freezing medium as indicated above.  The problem comes when we want to use these cells after storage.  All the cells are dead and there are no viable ones despite storage at the appropriate density and having been stored at greater than 90% viability.   What and where could be the problem?  Our thawing procedure is that we take the cells out of the liquid N₂ and thaw rapidly by swirling in a 37^oC water bath, add media, centrifuge and then grow them in a T25 container.   Could DMSO be the problem as we are not using cell-culture tested DMSO?

 Hi

DMSO concentration is vey important while cryopreserving cells. Moreover, A rapid thawing method ensures better results.
In your case,
You should always use the Cell culture grade DMSO and other reagents.
what is the final DMSO concentration after you dilute  cyropreserved cells following the thawing? The Final conc. of DMSO should not exceed 0.25%.
HAve you used gradual cooling method for cryopreservation or have rapidly cooled cells in liquid Ntrogen directly? Former is a better method.
Finally, HAve you optimized cryopreservation with 10 % DMSO ? The concentration varies with different cell types. For e.g rat primary granulosa cells show better results at 20% concentration.

All the best.

For a conservative cryopreservation protocol cool the cells (1-5e6/vial) slowly, meaning several hrs at -20o, then overnight at -80oC and the next day into nitrogen. For thawing, just put them in a floating rack in the waterbath (no swirling) and plate them (no centrifugation) in growth medium. Let them attach, usually overnight, then change the medium.

And use TC grade DMSO at 10 or 20% as the first replier suggests.