Makashova O. Influence of antioxidants on the state of cord blood nucleated cells during cryopreservation with cryoprotectant dimethylsulfoxide

The research subject was the effect of endocellular cryoprotectant DMSO and different antioxidants (glutathione, N-acetyl-L-cysteine, ascorbic acid) and low temperatures on structural and functional properties of nucleated, among them hematopoietic progenitor cells and human cord blood ones. The research aim was to determine the integrity, viability and the number of human cord blood nucleated cells with an excessive content of reactive oxygen species, as well as to study their apoptosis development during cryopreservation in the solutions, contained DMSO and substances with antioxidant properties. Research methods were as follows: light microscopy; flow cytometry with using fluorescently labelled monoclonal antibodies (CD45 FITC, CD34 PE, CD45 PE) and dyes (7-AAD, AnnexinV FITC, DCFH2-DA); isolation of cord blood nucleated cells by means of polyglucin; cryopreservation; statistical techniques. For the first time it was established the fact that the introduction into the DMSO-contained cryopreservation medium of the antioxidants of ascorbic acid, N-acetyl-L-cysteine or glutathione in efficient concentrations enabled to significantly increase the number of viable cord blood cells after freeze-thawing and to preserve a great number of undamaged post-thaw nucleated cells, and when transferring them into the conditions, simulating the physiological ones, in comparison with the samples, cryopreserved with no antioxidant supplement. In this case, the most pronounced effect was observed in the samples, cryopreserved with glutathione (1 and 3 mM). For the first time, the use of antioxidants in efficient concentrations during cryopreservation of nucleated cells was demonstrated to reduce the efficient concentration of DMSO from 10 down to 5%, thereby providing the decrease of toxic effect on cells. Herewith the integrity and viability remained at the level of the samples, cryopreserved with 7.5 and 10% DMSO with no antioxidant use.