Death of Cells After Defrosting - MDBK cells not surviving (Mar/20/2006 )

I am having an unusal problem with my MDBK cells. I received a vial for ATCC last October and passage 3 times prior to freexing down several vials according to recommendations provided by ATCC (95% Growth Media (which contains 10% FBS), 5% DMSO). I thought the serum and DMSO concentrations low from previous work with different cell lines but ATCC recommended this cryopreservation media. I placed the vials into a stryofoam container at -80C for 16 hours then transfered to liquid nitrogen tanks. Recently I needed to use the cells and removed a vial from liquid nitrogen, defrosted in 37C water bath quickly, diluted into 10 ml of prewarmed growth media and placed into T-75 flask. By late afternoon cells were looking great, plated wonderfully and flattened. I changed the media and placed back into the incubator. I gave them 24 hours before I checked and discovered that a majority of the cells had rounded up and lifted from the flask.
I have gone through several vials trying to identify the problem, used various dilutions of cells, used different flasks, made fresh media, and even altered the media (more serum and nutrients), double checked the incubator temp and CO2. Used Trypan Blue to check viability (77.5%) and contacted ATCC (no help). I believe it was the freeze process and nothing is going to rescue the cells, but wanted to exhaust all resources before I order more cells. Any suggestions would be greatly appreciated.

L. Rispoli

might be a dumb question...is your water pan in the incubator empty?

Although this doesn't seem to be your problem, but you do not wash your cells after thawing and thus leave the DMSO in the flask? Some colleague of mine did this with poor survival rates, then switched to a protocol with an extra wash step and her survival rate increased (though it is weird that your cells were first looking great).