5% dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO

Abstract

Background: Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system.

Study design and methods: We compared a lower DMSO concentration (5%) containing pentastarch with 10% DMSO and performed cell viability assay, colony-forming units (CFUs), and transplantation of CB cells in NOD/SCID IL2Rγ(null) mice.

Results: CB cells in 5% DMSO/pentastarch had similar CD34+, CD3+, and CD19+ cell percentages after thawing as fresh CB cells. CB cells in 5% DMSO/pentastarch had higher viability (83.3±9.23%) than those frozen in 10% DMSO (75.3±11.0%, p<0.05). We monitored cell viability postthaw every 30 minutes. The mean loss in the first 30 minutes was less in the 5% DMSO/pentastarch group. At the end of 3 hours, the viability decreased by a mean of 7.75% for the 5% DMSO/pentastarch and 17.5% for the 10% DMSO groups. CFUs were similar between the two cryopreserved groups. Frozen CB cells engrafted equally well in IL2Rγ(null) mice compared to fresh CB cells up to 24 weeks, and CB cells frozen in 5% DMSO/pentastarch engrafted better than those in 10% DMSO.

Conclusion: Our data indicate that the lower DMSO concentration with pentastarch represents an improvement in the CB cryopreservation process and could have wider clinical application as an alternate freezing medium over 10% DMSO.

Fig. 1.

Cell viability after thawing. CB samples (n = 4) cryopreserved in (○) 10% DMSO or (■) 5% DMSO/pentastarch were thawed and maintained at room temperature. A small aliquot was sampled every 30 minutes and analyzed for 7-AAD by flow cytometry. The percentage of viable cells (Y-axis) is presented with respect to time postthaw (X-axis). The error bars represent standard errors of the mean. There was a significant difference (p < 0.05) between 10% DMSO and 5% DMSO/pentastarch at time points 30 minutes and onward.

Fig. 2.

Percentage of human cell engraftment in NOD/SCID ILZRγ^null mice. CB MNCs (2 × 10⁷ cells per mouse, top) or CD34-se1ected cells (CD34+, 1 × 10⁶ per mouse, bottom) were infused (□, ○) fresh or (■, ●) after being frozen in 10% DMSO. Peripheral blood samples were assayed for human cells periodically. There were five mice per group; error bars represent standard error of the means.

Fig. 3.

Comparison of two freezing methods. Since most CB units are stored unselected, we infused CB MNCs fresh (■, 2 × 10⁷, n = 6), after being frozen with 10% DMSO (□, 2 × 10⁷ n = 7), or after 5% DMSO/pentastarch (, 2 × 10⁷ n = 6). The mean percentages of human CD45+ cells are plotted with respect to time after transplant. When t test was used to compare the two freezing methods, 5% DMSO/pentastarch had higher human cell engraftment at 6 weeks (p = 0.027, oneway ANOVA) and 12 weeks (p = 0.042, one-way ANOVA).