We are developing a protocol to isolate CD34+ cells from frozen bone marrow. We have tried thawing the bone marrow, running it on a SepMate15, and then using MACS CD34+ MicroBead kit, but we get very little separation with the SepMat15 tubes, leading to little to no CD34+ cell recovery. Does anyone have any suggestions on how to improve separation using frozen bone marrow or an alternative method?
Hi Michael,
I think that SepMate tubes are optimized for peripheral blood rather than bone marrow. Also, they probably work best with fresh samples. I would recommend comparing them in parallel with standard ficoll layering in 50ml falcon tubes and long centrifugation 300 x g, 30', deceleration set to 0.
I have done many CD34+ cells isolations with frozen cord blood and a few with bone marrow samples as well. Frozen samples have the advantage of being red-cell depleted - the DMSO causes red cell lysis - but have a nasty tendency to clump badly, leading to significant cell losses. I have greatly improved my recoveries using Miltenyi's Tyto Running Buffer, which is formulated to prevent clumping. I get good recoveries and I am able to isolate CD34+ cells by MACS.
The Tyto running buffer is expensive - over $1 per mL. What I do is thaw the sample, dilute with Hank's Balanced Salt Solution, spin down, resuspend the sample in Tyto buffer, spin again, and resuspend in Tyto buffer for a cell count. I run the MACS column in Tyto buffer - it does not interfere with the cell isolation - either positive selections or negative depletions. Once you have the selected fraction, you can go forward from there.
I have also used the Tyto running buffer for thawing peripheral blood stem cell products prior to FACS analysis and colony-forming assays.
My samples were cryopreserved in either 10% DMSO in FBS or in Pentastarch - described by Pat Stiff - which is 5% DMSO final concentration.
Dimitra Zacharaki and Gary Lee Gilmore thank you very much for your answers! If you have any other suggestions, please let me know