Effect of Various Concentrations of Dimethyl Sulfoxide (DMSO) on Cell Recovery and Clonogenic Potential of Cryopreserved Peripheral Blood Hematopoietic Progenitors

Hematopoietic stem cells mobilized to peripheral blood and collected by leukapheresis are predominantly used for autologous transplantation. Prior to cryopreservation the cells need to be processed including the addition of DMSO (dimethyl sulfoxide), which enhance cell survival, but is potentially toxic to stem cell recipient. The most commonly used concentration of DMSO is 10%. The goal of our study was to test if the concentration may be reduced without negative impact on cell recovery and clonogenicity.

Samples were prospectively collected from 12 patients with lymphomas mobilized with chemotherapy combined with G-CSF. Small volumes (2–3 ml) of cell suspensions obtained from the leukapheresis product were divided into 4 parts placed in separate small vials, each containing different cryoprotective mixture - with 10%, 7.5%, 5% and 2.5% DMSO. The final volume of cell suspensions equaling 1 ml, the cell concentration (0.8–1 × 10⁸ /ml) and the proportions of human albumin and plasma were the same in all vials. The cells were frozen in IceCube, using a computer controlled cooling program and stored in liquid nitrogen for 2 weeks – 4 months. The quality of cryoprotective mixtures was evaluated by cell recovery and clonogenic potential. The recovery was determined by comparing number of living cells before and after cryopreservation, using trypan blue staining. Clonogenic potential was carried out by colony forming unit (CFU) assays. Depending on CD34⁺ percentage, 5 or 10 × 10³ living cells were plated (in triplicates) in medium MethoCult and cultured for 14 days.

The median recovery of nucleated cells for 10% DMSO was 62.4% (range 41.2–86.8) and was significantly higher compared to 7.5% DMSO (54.9%, 41.2–89.1; p=0.04), 5% DMSO (49.2%, 28.1–69.8; p=0.002) and 2.5% DMSO (37.2%, 19.3–54.3; p=0.002). The number of CFUs calculated per 100 000 cryopreserved cells did not differ significantly according to DMSO concentration: 217 (14–1795) for 10% DMSO, 225 (27–2718) for 7.5% DMSO, 196 (26–2761) for 5% DMSO, and 178 (14–2208) for 2.5% DMSO. Neither cell recover nor clonogenic potential correlated with the percentage of CD34+ cells in the leukapheresis product.

Reduction of DMSO concentration to equal or below 7.5% is associated with impaired recovery of nucleated cells after cryopreservation. However, it does not appear to negatively affect clonogenic potential of leukapheresis product, suggesting relative resistance of hematopoietic progenitor cells. Therefore, considering potential toxicity of DMSO to stem cell recipient, its lower concentrations may be clinically beneficial. This hypothesis requires prospective verification in a setting of autologous transplantation.

No relevant conflicts of interest to declare.