We have some frozen cell-lines in freezing medium (10%DMSO in FBS), which we want to fix using 10% NBF and eventually make FFPE blocks with. Would it be ok to just thaw them rapidly and wash them with PBS by spinning (to get rid of the freezing medium) and then resuspend the pellets in NBF to be fixed? Will this damage the cells?
We don't want to culture them prior to fixing.
Thanks!
Dear Dr. Maral Tabrizi
The protocol that you have mentioned to fix cryopreserved cells using 10%NBF may be damaging to cells. The cells will undergo a lot of stress as you will be subjecting them to many treatments like thawing, washing, centrifugation and fixation within a short period without giving the cells enough time to recover. This is going to be stressful to the cells.
I would recommend that you first thaw the cells by a quick thaw method, culture the cells for 2-3 passages, trypsinize the cells on confluency, wash once with PBS, and resuspended in approximately 150-200 μl of 10% neutral buffered formalin (NBF).
You may transfer the resuspended cells in a microcentrifuge tube with solidified 2% agarose prefilled in the tapered portion of the bottom, and spin down at 540 ×g for 5 minutes. The resulting supernatant may be removed, and you may add fresh 10% NBF without disturbing the pellet. The microcentrifuge tube may be spun at 810 ×g for 5 minutes, and the resulting cell plug may be fixed in formalin for 48 hours at room temperature by submerging the microcentrifuge tube into a 15ml conical tube containing 10% NBF.
After fixation, the microcentrifuge tube may be transferred to a new 15ml conical tube with PBS and stored at 4°C prior to processing and embedding in paraffin blocks.
I feel this method would be more appropriate than fixing the cryopreserved cells.
Best.
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