Dear friends,
I am planning to cryopreserve and generate a HEK293T cell bank, but I have a few doubts on how to achieve best results. I am using "Corning® CoolCell® FTS30 container" for the ultrafreezing step prior to transferring to the liquid N2 tank.
1. Should I chill the cells prior to freezing (i.e. leave them to reach 0-4ºC in the fridge after aliquoting the final cell suspension in freezing medium (FBS, 10% DMSO))?
2. Should I chill the CoolCell between 0-4ºC before transferring the cryovials into it? If so, should the vials be pre-chilled also at 0-4ºC or be at RT?
3. How long exactly should the cells stay inside the ultrafreezer before going into the tank?
Any other tip or amplification would be very much appreciated.
Thanks a lot.
To keep HEK293 cells, use a glucose rich growth media such as Dulbeccos modified eagle medium (DMEM) or Eagles minimum essential medium (EMEM); this can be supplemented with Fetal bovine serum (FBS) to a concentration of 10%. The media can then be dispensed to cryovials at room temperature, which can then be placed in the freezer, where it can freeze to required temperature; either 0-4 or less degree centigrade.
I use these containers all the time with good results. I always add the tubes to the containers at room temperature, transfer straight into the -80oC freezer and then in to liquid nitrogen the next day.