Maslova O. Morphofunctional characteristics of mesenchymal cells of the umbilical cord in the modified conditions of culturing

The purpose of the thesis was to obtain mesenchymal cells from umbilical cord matrix, to optimize approaches for culturing mesenchymal cells from human umbilical matrix and to evaluate their morphofunctional characteristics in passages. To achieve this goal the following objectives were identified: to analyze the effectiveness of obtaining cultures of mesenchymal cells that meet the criteria of MSC approved by the International Society for Cellular Therapy (2006) from human umbilical cord; to develop criteria for evaluation of morphometric characteristics of human umbilical cord; to develop individualized protocol for obtaining MSCs from human umbilical cord based on its morphometric parameters; to determine the morphological features of cultured cells at different passages; to study the effect of synthetic protein kinase inhibitors on the viability and morphological characteristics of MSCs. The subject of work: human umbilical cord as a source of cell material and culture of mesenchymal cells that were obtained from the umbilical cord matrix. Modified classical methods of cells' isolation and cultivation, induction of chondrogenesis; cytological staining techniques, followed by application of light, interference and confocal microscopy; FACS analysis, modern statistical methods were used. The research allowed to determine the best approach to isolation and culturing mesenchymal stromal cells from human umbilical cord, to describe morphofunctional features of the obtained cell culture and to contribute to the preparation methods of the potential use of these cells in cell therapy. The following conclusions were made: We recommend choosing the most convenient method of processing the umbilical cords depending on their organometric parameters. Following the evaluation of the value of stromal-vascular rate (SVR), it should be decided whether to use the enzymatic or mechanical approaches or to combine them. Each cord sample has its own architectonics; so it is impossible to use one common method to isolate cells. Therefore cords with low SVR should be treated with the predominance of enzymatic approaches in contrast to cords with high SVR that should be treated using mechanical approaches. It has been shown that platelet rich plasma at a concentration of 10% serves as the best substitute for xenogenic components of the culture medium for MSCs. The effects of synthetic protein kinase inhibitors MI-1 and D-1 in various concentrations on the culture of MSCs at different passages were demonstrated. The changes in the morphological characteristics of human umbilical cord mesenchymal cells during long passaging were shown. Accordingly, we recommend using umbilical cord matrix cells at stages not later than the 2nd passage. The possibility of spontaneous formation of cell conglomerates ("mesenspheres"), with preserved functional properties of mesenchymal cells in cultures derived from some samples of umbilical cords in standard culture conditions can serve as an argument in favor for heterogeneity of umbilical cords.