We have stored and used frozen mesenchymal stromal cells from humans and dogs at our lab. They are frozen also at 10% DMSO concentration. Further we also regularily use these cells for qPCR and flow cytometry. These cells are isolated from bone marrow. So are likely similar.

I would highly recommend thawing and washing away the DMSO before using these cells for downstream processes. This is particularily important for qPCR, since DMSO has a clear effect on qPCR reaction kinetics (you can even use very low concentrations of DMSO to optimize the reaction efficiency; i think it modulates DNA melting/annealing).

For flow cytometry you also want to get rid of DMSO, for several reasons. First, if you plan any immunophenotyping, DMSO can also change reaction kinetics of antibody-antigen complex formation. Second, DMSO is toxic at 10% after 15-30 minutes at temps greater than several degree celcius, which would result in cell death, lysis, and fragmentation, not great for getting enough events for good FC analysis. Large cells fragments call also bind antibodies and probes! Third, DMSO might interact with the peristaltic tubing in the cytometer, degrading it.

In general as well, thawing any cells from -150C in DMSO will cause some cell death and lysis. You want to remove these cellular debris from your sample, which can only be done by washing after thawing. Lots of aggregated debris will clog you cytometry filter.

So wash away. As to how we wash our cells after thawing, here is a basic process.

1. Create a thawing solution. We use whatever media the cells were frozen in (in our case typically DMEM), with 10% FBS, and 1 ug/mL of DNase. Create enough that you can dilute your frozen cell sample more than 10X (to reduce DMSO concentration to less than 1%). Warm this media to 37C.

2. Have a water bath ready at 37C. Prepare a tube of warmed thawing media for each individual/test sample you want (or 2 tubes per cryovial if you want to split cells for both cytometry and RNA isolation). Add enough media to the tube to dilute the DMSO to less than 1% (i.e 1 mL cryovial needs 10 mL media). I prefer to use 15 or 50 mL falcon tubes; the cells seem to pellet much better in these than in eppendorfs. Also, given standard cryovial volumes you will typically need more than a few mL of media anyways.

3. Bring up several cryovials on dry ice (not normal ice! maintain -80C until transfering cryovial to warmed waterbath). Take one cryovial and submerge until water almost reached cap of tube. Gently swirl the vial until a small ice crystal remains. Remove vial from water bath and spray with ethanol, wipe down.

4. Transfer contents of cryovial to the warm media in falcon tube. Do this as gently as possible. Rinse cyrovial with some of this media after transfer, and transfer the rinse media back to falcon tube as well. Gently invert the falcon tube several times. Repeat for other cryovials.

Note: since DMSO become toxic fairly rapidly after thawing, i prefer to thaw my cryovials in blocks of 4.

5. Centrifuge falcons at several hunderd xg for 5 minutes. (200 - 500xg seems ok, but you should test). I prefer acceleration and break at 9 and 5 respectively, which creates an easier to respusepend pellet, but you might lose more cells that 9 and 9. Aspirate SN by tipping the tube, dont let aspirator get too close to pellet, leave around 100 uL of SN above pellet.

6. At this point, you can directly resuspend the cell pellet for RNA in trizol or RNA isolation kit buffer. Any RNA kit for cells should work at this point, i prefer column based kits because they are fast. We use Aurum Total RNA kit from BioRad, gives high quality RNA (minimal fragmentation and contamination). For the flow cytometry pellet, resuspend in buffer of choice, i.e. if you are doing immunophenotpying, resuspend in this buffer. Resuspend gently, either by flicking the pellet, or, using the largest pipette width you have (to reduce shear force), pipette cells up and down slowly until fully resuspended.

A BIG CAVEAT: This works for us with frozen MSC. However, how well your cells respond to thawing really depends also on HOW they were frozen. I would very much urge you to somehow test the above process to see it is optimal for your freezing process. I know you mentioned you have no test material. However, this means you have precious cellls which you are not expanding/culturing) and you want to jump right into using them??? This is very risky... I would urge you to purchase frozen bone marrow cells for the species you are using, im sure you can find some offered by cell banks, and test thawing. Or, purchase fresh cow bone marrow (i know its not mice or dog, which is likely what you used, but most mamilian cells respond similaraly on thawing), and isolate and freeze the bone marrow according to your labs protocols. Then optimize thawing given the protocol i described.

Good luck!

Thank you very much for a detailed answer Gion Fränkl :) Really appreciate it!

It is true that we do not have any test material. We do, however, have 4 aliquots of cells per rat. It really is precious material and I will consider buying some bone marrow cells, freeze them in the same way that was done during the animal experiment and try out the thawing procedure, as you suggested!

Again, thank you very much, it is very helpful :)