when a small frozen cell pellet remained I added dropwise pre-warmed media to the collection tube. I used the same viability/surface markers to stain for flow and the majority of cells (75%) appear to be dead. Can anyone suggest a freezing media or an alteration to my protocol in order to avoid the cell death after freeze/thawing? I used cryostor to avoid any animal serum to stop any immunological reactions. thaw them by putting them in 37C waterbath and when a small frozen cell pellet remained I added dropwise pre-warmed media to the collection tube. I used the same viability/surface markers to stain for flow and the majority of cells (75%) appear to be dead. Can anyone suggest a freezing media or an alteration to my protocol in order to avoid the cell death after freeze/thawing?
I would thaw your cells relatively quickly in a 37 degree water bath and just as they are thawing add them in a large volume (10x the frozen storage volume) of chilled (4 degree) media containing serum (something the cells will like). Then spin them down in a centrifuge, decant the media and then add whatever volume is needed of chilled media to resuspend the cells and count them. Keep the cells cold until they can be adjusted to their working concentration, then you can warm them up. The goal is to dilute the DMSO present in the Cryostor. 10% DMSO is quite toxic to cells at 37 degrees, and somewhat toxic at 4 degrees.
Of course your cell death could also be happening during the freezing procedure. In this case the cells should be chilled, concentrated, and added to the chilled cryostor and immediately frozen. If you have access to a LN2 programmed freezer this would help, otherwise get them into a -80 overnight and then into LN2.
Again the goal is to keep the cells cold while in the presence of the unfrozen cryostor. If you don't need to use cryostore then freezing in 90% serum, 10% DMSO works well too.
You can also replace serum with a 20% albumin, it is not the same but works well. You can use 10% DMSO in medium supplemented with 10 or 20 % albumin. Cryostore should work well either, are you performing a controlled freezing?
I can also add that if your sample has high myeloid or granulocyte cells, viability is going to be low. Those cells can have very low viability after cryopreservation, but lymphocytes from that very sample have over 70% viability.
Hope it helps!
I agree with James.
Also I think your problem is during the freezing procedure.
I worked with cryostor and I didn't have any problem with that.
I think you need to freeze/thaw your cells as fast as posibble
Thank you all for the response! I agree that it is the freezing method and after keepomg evertthing cold I get around 70% viability.
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