ANALYSIS OF THE STRUCTURAL AND FUNCTIONAL STATE OF HUMAN CORD BLOOD HEMATOPOIETIC PROGENITOR CELLS AFTER CRYOPRESERVATION WITH DMSO AND ANTIOXIDANTS AND TRANSFER TO CONDITIONS CLOSE TO PHYSIOLOGICAL
About the author:
Makashova O. Ye., Zubova O. L., Zubov P. M., Babijchuk L. A.
Type of article:
The use of cord blood (CB) hematopoietic progenitor cells (HPCs) for the past 15 years has been firmly establishing in practical medicine as an effective treatment for diseases of various origins. In this regard, it remains relevant for the development of 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 that during cryopreservation HPCs are subjected to a significant stress, resulting in the decrease of the antioxidant system work, disruption of the membrane structure and the glutathione release from the organelles, which leads to an increase in the level of ROS in cells after transferring them to the bloodstream and the development of lipid peroxidation. In addition, there are suggestions that in the initial period after warming, the level of lipid peroxidation is limited by the structural antioxidants of the glutathione peroxidase system. In this regard, to increase the efficiency of cryopreservation of cord blood preparations, an important task is not only to evaluate them immediately after thawing, but also to determine the delayed survival of cells after transport them to conditions close to physiological. The addition of antioxidants to the cryoprotective medium can improve the preservation rate and viability of HPCs both immediately after cryopreservation and after transfer to conditions that are close to physiological. Based on this, the aim of the work was to analysis of the structural and functional state of human cord blood hematopoietic progenitor cells after cryopreservation with cryoprotectant DMSO and antioxidants and transfer to conditions close to physiological The efficacy of ascorbic acid, N-acetyl-L-cysteine and glutathione application in combination with DMSO in cryopreservation of HPCs 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 HPCs decreases on 20% in the samples, cryopreserved with 7.5-10% DMSO. 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 HPCs 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 HPCs up to 70% 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 80% of the HPCs. This may be due to the fact that glutathione reduced the number of cells with excess content of ROS after transferring the cells to conditions that are close to physiological.
cord blood hematopoietic progenitor cells, cryopreservation, DMSO, ascorbic acid, N-acetyl-Lcysteine, glutathione
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Publication of the article:
«Bulletin of problems biology and medicine» Issue 1 Part 1 (142), 2018 year, 384-388 pages, index UDK 612.649.011.87.014.3:615.014.41:547.569.2