Hello everyone,

In my lab, we isolate mononuclear cells (MNC) from umbilical cord blood (UCB) using density-gradient isolation (Ficoll-Paque), use NH4Cl to remove erythrocytes, and then we cryopreservate them in DMEM+10%FBS and 10% DMSO.

In the last two years, we have been having very low recoveries (10%-30%) after thawing and high levels of apoptotic cells (after Annexin V assay). Previously this was about 50%-60%. The only thing that we changed was the anticoagulant in the collection bags from CPDA1 to CPD.

We tried almost everything and we cannot find ways to increase these recoveries. We often observe very high erythrocyte contamination in the buffy coat. We already tried to cryopreservate in FBS+DMSO, changing the centrifugation settings (400g, 30 min, breaks off for density-gradient).

Have you ever had this problem? Do you know any solution?

TIA