I used to freeze A-498 and CAKI-2 cell line at the same time with the same reagents (90 % FBS and 10 % DMSO), store the vial at -80 °C in polystyrene box for 24 h, after then I transfer the vials to liquid nitrogen. After a few days when I check the cells I experience that the CAKI-2 cells do not attach to the flask, they are floating in the media. Any suggestion on what am I doing wrong?
Thank you in advance for the answers
Provided you worked quickly once you introduced dmso to your cells, one thing i notice (or dont notice) is whether or not you are storing vials in -80 utilizing isopropanol or an isopropanol-containing vessel. This is to allow proper freezing of the cells (-1c degree/minute). I have never frozen down cells without these vessels (https://www.thermofisher.com/order/catalog/product/5100-0001?ca&en); thus if you are not, this could cause your cells to die?
Can anyone comment about freezing cells successfully WITHOUT these alcohol containing vessels?
Also minor things (you may already do) such as when you thaw, thaw as quickly as possible (in 37c water bath) and get them pelleted again asap so you can add prewarmed fresh media which does not have any dmso in it.
Hi, I used to do kind of a russian doll with styrofoam boxes, to allow the cells to freeze gradually before transferring into the liquid nitrogen. Now I use a single styrofoam box and they all do well. But, depending on the cell line, the survival after thawing depends on the number of cell too. Some I put 2 millions cells in the vials, other up to 4 millions.
I agree with Aaron regarding the timing, you have to act quickly after adding the DMSO and when I thaw them in the 37C bath, I always resuspend in fresh media, centrifugate and resuspend again in fresh media to eliminate any trace of DMSO.
Also, I found that some cells prefers to be started in smaller flasks and when the reach confluence I transfer them in a bigger one.
Hope that helps!
Are you mixing the cell lines? They require different growth mediums. The medium should be supplemented with 10% FBS and only 5% (v/v) DMSO. Also, as listed in the other answers, it's very important that it freezes over time. Flash freezing may cause morphological changes or kill the cell lines. Consider using a Mr. Frosty container with isopropyl alcohol, which will allow it to freeze at roughly 1 degree C per minute. Also, reiterating the answers listed above, please double check your thawing protocol.
Attached are the info sheets for both cell lines.
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