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We studied the influence of sucrose applied in combination with different concentrations of penetrating cryoprotectants (DMSO, ethylene glycol, and glycerol) on the efficiency of cryopreservation of umbilical cord-derived multipotent stromal cells (MSC). The results indicate that these cells can be cryopreserved with the use of 5-10% DMSO or ethylene glycol with equal efficiency; addition of 0.2 M sucrose does not affect cell survival after thawing. The efficiency of glycerol as a cryoprotectant increases with increasing its concentration from 5 to 10%, but remains significantly lower than the efficiency of DMSO or ethylene glycol. Addition of sucrose to a final concentration of 0.2 M increases the efficiency of glycerol. The efficiency of combination of 10% glycerol and sucrose was comparable with that of combinations of DMSO and ethylene glycol with sucrose. The mechanism of the observed enhancement is apparently related to the influence of sucrose on the dynamic properties of the lipid membranes and facilitation of glycerol diffusion into the cells.

The mechanisms of proangiogenic activity of multipotent stromal cells from human umbilical cord were analyzed in vitro. The absence of secreted forms of proangiogenic growth factor VEGF-A in the culture medium conditioned by umbilical cord-derived multipotent stromal cells was shown by ELISA. However, the possibility of paracrine stimulation of cell proliferation, mobility, and directed migration of endothelial EA.hy926 cells was demonstrated by using MTT test, Transwell system, and monolayer wound modeling. The capacity of multipotent stromal cells to acquire the phenotype of endothelium-like cells was analyzed using differentiation media of three types. It was found that VEGF-A is an essential but not sufficient inductor of differentiation of umbilical cord-derived multipotent stromal cells into CD31+ cells.

Human umbilical cord represents a source of multipotent stromal cells of a supreme therapeutic potential. The cells can be isolated from either fresh or cryopreserved umbilical cord tissues. DMSO is a cryoprotectant most commonly used for preservation of umbilical cord tissues; however, cyto- and genotoxicity of this compound is evident and well documented. In the present study we performed successful cryopreservation of the umbilical cord tissue using other cryoprotectants: propylene glycol, ethylene glycol, and glycerol. Of these, 1.5 M ethylene glycol and 20% glycerol turned out to be the best in terms of the preservation of living cells within the frozen tissue, early onset of migration of these cells out of the thawed explants, and overall efficacy of multipotent stromal cell isolation. Cryobanking of tissues can improve availability of multiple cell products for medical purposes and promote the development of personalized medicine.

Short-term cell culturing on basement membrane matrix is a common and very convenient in vitro model of angiogenesis. We studied the possibility of interaction of multipotent stromal cells from the umbilical cord and Ea.hy926 endothelial cells on this model at the early and late periods of the experiment. Multipotent stromal cells alone and in combination with endothelial cells formed an unstable tubular network. Clusters formed after its disassembling later became the sprouting centers in co-culture of the two cell types, but not in pure culture of multipotent stromal cells. Multipotent stromal cells with CD31+ phenotype constitute the structural basis of newly formed stable 3D capillary-like network. Prolongation of the time of culturing and combination of the two in vitro models of angiogenesis (tubulogenesis and sprouting) allowed more complete assessment of the angiogenic potential of multipotent stromal cells.

Intrasplenic allogeneic transplantation of multipotent stromal cells from the umbilical cord stimulates hepatocyte proliferation and promotes recovery of liver weight in rats after subtotal resection (80% organ weight). It can be hypothesized that this effect of multipotent stromal cells is due to more rapid recovery of the number of mitochondria and normalization of mitochondrial function of liver hepatocytes.

The role of the lungs and kidneys in liver regeneration after subtotal hepatectomy was studied on a rat model. It was found that production of hepatocyte growth factor (HGF) in the lungs and kidneys and expression of cytokine genes Il1b, Il6, Il10, and tnfa significantly increased. Analysis of the dynamics of lung macrophage population showed that accumulation of HGF and the increase in the expression of cytokine genes in the lungs were accompanied by simultaneous increase in the number of CD68+ cells, which attested to the leading role of macrophages in activation of HGF synthesis in the lungs. Macrophage content in the kidneys after subtotal hepatectomy did not increase.

The proliferation and death of hemopoietic cells in the regenerating liver of 17-day outbred albino rat fetuses were studied during 2 days after resection of 20% of the organ. The mitotic index of hemopoietic cells in the resected liver increased signifi cantly 24 h after the operation in comparison with the control fetuses. No increase in the counts of apoptotic hemopoietic cells was detected in the regenerating liver. Hence, resection of 20% of the liver in rat fetuses stimulated the proliferation of hemopoietic cells and did not stimulate their apoptosis.

Expression of il1b, il6, il10, tnfa, hgf, tgfb, vegf, and fgf2 genes in the lungs and kidneys was examined on rat model of liver regeneration after subtotal hepatectomy. Enhanced expression of il6, il10, tnfa, hgf, and fgf2 genes was detected at the early terms after 80% liver resection.

In the liver of rats subjected to subtotal liver resection (80% organ weight), the expression of sox9 gene and SOX9 protein content increased and cells with hepatocyte morphology expressing SOX9 appeared; the proportion of cells expressing cytokeratin-19 also increased. Based on these data, we cannot completely exclude the involvement of resident progenitor cells and hepatocyte reprogramming in liver regeneration after subtotal resection, however, the contribution of these processes seems to be insignificant. The leading mechanism of liver mass recovery after subtotal resection is proliferation of hepatocytes.

We studied hepatocyte proliferation in the regenerating liver of 17-day fetuses of outbred albino rats. The animals were sacrificed every 3 h over 2 days after resection of 20% liver. The number of mitoses beyond the zone of injury increased sharply 12 and 24 h after resection. The hepatocyte mitotic index in the perifocal zone did not surpass the mitotic index in regions distant from the focus of injury during any of the studied periods. No circadian rhythm of hepatocyte mitotic activity was detected for resected or intact fetal liver. The injury caused virtually no changes in hepatocyte mitosis phase ratio in the operated compared to intact liver, which attested to stable course of mitosis. The weight of fetal liver recovered at the expense of enhanced mitotic activity of hepatocytes in the entire liver.

A reproducible model of fetal liver regeneration was created. Resection of 20% liver was carried out in rat fetuses on day 17 of prenatal development. The organ weight was restored after 2 days at the expense of an increase in hepatocyte mitotic activity; cell hypertrophy was minor. After recovery, the cell composition of the operated liver did not differ from the control, i.e. the regeneration was organotypical.