The Effect of Antioxidants on the State of Cryopreserved Human Cord Blood Nucleated Cells after Transferring them to Conditions Close to Physiological

The use of cord blood (CB) hematopoietic progenitor cells for the past 15 years has been firmly established in practical medicine as an effective treatment for diseases of various origins. In this regard, it remains relevant for developing the protocols for cells storage at low temperature. Most of them are based on the use of penetrating cryoprotectant DMSO in effective concentrations. However, they do not take into account the fact that during cryopreservation cord blood nucleated cells are subjected to a significant stress, resulting in the accumulation of high concentration of reactive oxygen species (ROS). The literature analysis evidences that ROS can initiate the development of apoptosis and cell death both before and after cryopreservation. Therefore, the addition of antioxidants to the cryoprotective medium can improve the preservation rate and viability of cord blood nucleated cells both immediately after cryopreservation and after transferring them to conditions close to physiological. There are assumptions that in the process of cryopreservation cells are subjected of certain signals that make them "susceptible" to the development of apoptosis, but not initiate its development at this moment. Therefore, an important task for scientists is not only to conduct their evaluation immediately after thawing, but also to determine the delayed survival of cells after transferring them to conditions close to physiological. Based on this, the purpose of the work was to evaluate the effectiveness of cryopreservation of cord blood nucleated cells (CBNCs) that were cryopreserved in protective solutions containing various concentrations of DMSO and antioxidants after transferring to conditions close to physiological. The efficacy of application of ascorbic acid, N-acetyl-L-cysteine and glutathione in combination with DMSO in cryopreservation of CBNCs has been evaluated. It has been shown that in the process of cryopreservation with DMSO and after transferring to conditions that are close to physiological, the preservation and viability of CBNCs decreased. 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 CBNCs 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 CBNCs up to 65% in comparison to the samples without adding these 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 75% of the CBNCs. This may be due to the fact that glutathione and acetylcysteine reduced the number of cells with excess content of ROS after transferring the cells to conditions close to physiological.