Cryopreservation of Cord Blood Hematopoietic Progenitor Cells in Cryoprotective Media Containing Various Concentrations of DMSO and Antioxidants

The use of hematopoietic progenitor cells (HPC) for the past 15 years has been firmly established in practical medicine as an effective treatment for diseases of various origins. Nowadays, one of the sources for obtaining hematopoietic progenitor cells is human cord blood (CB). Cord blood has become one of the most popular sources of HPC due to its unique properties, relative simplicity and security of procurement. The number of transplantations is increased every year, and the indications for this type of therapy are expanding. In this regard, it remains as an urgent for the development of protocols for cells' storage at low temperature. Adding antioxidants to the cryoprotective medium may enhance preservation and viability of cord blood HPC after cryopreservation and result in clinical efficacy improvement. The aim of this work is to assess the efficacy of antioxidants in combination with different concentrations of dimethylsulfoxide (DMSO) for cryopreservation of HPC. The efficacy of application of ascorbic acid, N-acetyl-L-cysteine and glutathione in combination with DMSO in cryopreservation of cord blood HPC has been evaluated. It has been shown that in the process of cryopreservation with DMSO, the preservation and viability of HPC decreases. One of the reasons for this may be the accumulation of reactive oxygen species (ROS) in cells during freezing. The addition of antioxidants to the cryoprotective medium can significantly increase the stability of the HPC against the effects of cryopreservation factors and improve the preservation and viability indices. A comparative analysis of antioxidants revealed that ascorbic acid at concentrations of 0.1 and 0.15 mM and N-acetyl-L-cysteine (10 and 15 mM), in combination with 7.5% and 10% DMSO, increased the number of preserved viable HPC by 15% in comparison to the samples without adding the antioxidants. Addition of glutathione at a concentration of 1 and 3 mM to cryoprotective medium with 7.5% and 10% DMSO was able to maintain a viable state of up to 90% of the HPC, unlike the two other antioxidants that ensured the viability of 80% of the cells. It should be noted that the results of cryopreservation of HPC in solutions containing 5% DMSO and the above-mentioned effective concentrations of antioxidants were at the level of data obtained with optimal concentrations of DMSO (7.5 and 10%) without the use of antioxidants, which allowed decreasing the concentration of DMSO when there is cryopreserving the HPC.