Mykhailova O. Structural and functional state of cord blood nucleated cells at different stages of cryopreservation

The research object was the influence of cryoprotectants with different mechanism of action (penetrating in the cells DMSO and non-penetrating PEG-1500) and low temperatures on the structural and functional properties of the nucleated, including hematopoietic stem/precursor, human cord blood cells isolated by different methods. The research aim was a complex assessment of the structural and functional state as well as their viability of different populations of human cord blood nucleated cells, including hematopoietic stem/precursor cells, depending on the isolation methods from whole blood, as well as under the influence of cryoprotectants with different mechanism of action and low temperatures. Research methods were light microscopy, flow cytometry using a monoclonal antibody, Annexin V-FITC and fluorescent dyes DCFH2-DA and 7AAD, centrifugation, cryopreservation and the methods of mathematical modeling and statistical analysis of the results. Equipment comprised software freezer "Cryoson" (Germany), flow cytometry FACS Calibur (BD, USA). The complex assessment of structural and functional state of different populations of cord blood nucleated cells, including hematopoietic stem/precursor cells, at each stage of cryopreservation, and determination of the processes occurring in the cells under the influence of cryoprotectants with different mechanism of action and low temperatures were carried out for the first time. This study demonstrates the possibility for quantitative and qualitative preservation of the cellular composition in cell-based products cryopreserved under protection of 5% DMSO (after isolation in polyglucinum) and 10% PEG-1500 (after isolation by two-step centrifugation), i.e. more than 80% CD45+ - and 90% CD34+-cells in viable state. The research results have scientific and practical importance for the establishing of cord blood nucleated cells stores and their long-term storage in order to further clinical application. The results can be used for development of new and improvement of existing technologies of cell suspensions cryopreservation, as well as for professional advancement of specialists in different fields of biology and medicine.