Aim: to determinate the most effective liver cells and multipotent mesenchymal stromal cells of bone marrow (MMSC BM) ratio into implantable cell engineering constructions (CECs) used for chronic liver failure (CLF) correcting.Materials and methods. For creating liver CECs it was used a biopolymer implant – a composition of a heterogeneous collagen-containing gel (BMCG) (Sphero®GEL trademark) containing viable liver cells and MMSC BM in the following ratios – 1 : 1; 5 : 1 and 10 : 1 respectively. CECs with different ratios of liver cells and MMSC BM were implanted into liver of rats in which chronic liver failure (CLF), was modeled by using CCl4. The effectiveness of the regulatory effects of CECs (with different cell ratios) on regenerative processes in livers were assessed by using biochemical, morphological and morphometric methods at different periods after their implantation.Results. Corrective effect of CECs with different cell composition on biochemical and morphological parameters of livers at chronic liver failure was established. During studying the liver CECs with various cell ratios of liver cells and MMSC BM (1 : 1; 5 : 1 and 10 : 1 respectively), it was found that the most optimal ratio of cells into the CECs is 5 : 1, because at this ratio of cells, there were a more distinct normalization of the morphological and functional liver parameters within 365 days after modeling CLF and maintenance of the structural homeostasis into the CECs. Themselves, which allows predicting their long-term regulatory effect on the liver tissue in CLF and maintaining its normal structural and functional state.Conclusion. The effective correction of chronic liver failure can be carried out by using the implanted liver CECs, in which donor liver cells and MMSC BM where presented in ratios – 1 : 1; 5 : 1 and 10 : 1. But analysis of prolonged correction of liver morphological and functional parameters at CECs using it was allow to recommend the preferences using of CECs with ratio 5 : 1, because prolonged preservation of structural homeostasis into these CECs makes possible to prognosticate their prolonged regulatory action on the liver tissue at CLF, especially for recipients on a waiting list for liver transplantation.

Extensive liver resection (ELR), performed in a number of surgical operations, refers to a critical injury, which necessitates the improvement of methods of therapy of acute post-resection liver failure.Aim:to compare the effectiveness of stimulation of regenerative processes in the liver residue after ELR (60–70%) by intraperitoneal administration of lysed aspirate bone marrow cells (BMCs) and total RNA (tRNA) isolated from BMCs.Materials and methods.This work was performed on 175 rats-male Wistar breed 250–300 g, on 75 of which under the inhalation anesthesia it was reproduced the model of the ELR in three groups of experiments: group 1 – control (administration of isotonic solution after ELR), group 2 – in 3–5 hours after ELR the tRNA from BMCs was intraperitoneally injected at a dose of 30 μg/100 g, group 3 – in 3–5 hours after ELR BMCs was administered intraperitoneally at a dose of 30–35 × 106cells per rat. Comparative studies of the restorative processes in the liver after the ELR in the three groups were carried out by dynamic control of the mitotic activity of hepatocytes in the liver residue, cytolytic enzymes, total bilirubin and total serum protein, as well as the liver residue (mass) weight.Results.The tRNA from BMCs and BMCs in the indicated doses prevent the risk of the development of lethal outcomes, and also contribute to an earlier (by 10–14 days) normalization of the functional indices of hepatic homeostasis. However, the tRNA from BMCs, compared with BMCs, has a stronger stimulating effect on the recovery processes: it promotes earlier intensification of mitotic activity of hepatocytes and provides a higher rate of recovery of liver mass.Conclusion.For the induction of recovery processes in the liver residue after ELR, the preference should be given to the tRNA from BMCs.

Objective: to study the cellular mechanisms of activation of regenerative processes in the liver when using total RNA (tRNA) of bone marrow cells (BMCs) based on an extended liver resection (ELR) model. Materials and methods. Male Wistar rats (n = 80) with ELR model (70%) were divided into 2 groups: group 1 (control group) had a single saline injection, while group 2 (experimental group) received a single tRNA injection at a 30 μg/100 g dose of animal weight. The biochemical parameters of liver function and weight were monitored over time. Also monitored were microstructural changes in hepatocytes 48 hours after ELR by examining mitotic activity, caspase-9 expression and morphometric parameters. Results. It was found that in group 2, in comparison to group 1, there was faster normalization of biochemical parameters (by 10–14 days), a higher mitotic index of hepatocytes (23.45‰ versus 5.37‰), and initially sharper decrease and then faster recovery of liver mass (by 10–12 days versus 18–20 days). Both groups showed almost total expression of caspase-9, including in mitotically splitting hepatocytes. Group 1 demonstrated decreased values of morphometric parameters of single and binuclear cells, decreased number of binucleated hepatocytes and increased total density of hepatocytes as compared to the intact liver. Intraperitoneal administration of tRNA increased morphometric parameters of mononuclear hepatocytes, did not affect their number, but increased the area of the nuclei of binuclear hepatocytes as compared to the control group. Conclusion. The proven capability of cell-bone marrow total RNA to simultaneously support apoptosis in liver cells after ELR and induce mitotic activity indicates that tRNA can switch activated apoptosis to cell proliferation at the early phase of the regenerative process. This effect may be due to the presence of regulatory RNA molecules in tRNA, including numerous non-coding RNAs.

Shortage of donor organs for liver transplantation in the treatment of end-stage liver disease dictates the need to develop alternative methods that include technologies on tissue engineering and regenerative medicine. Objective: to study the ability of a tissue-specific matrix from decellularized human liver fragments (DHLF) to maintain adhesion and proliferation of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and HepG2 under static conditions and in a flow-through bioreactor. Materials and methods. Treatment with surfactants (SAS) – sodium dodecyl sulfate, Triton X-100 – followed by exposure to DNase was used for decellularization of human liver fragments (no more than 8 mm3). Biochemical screening included the determination of DNA quantity in the test samples. Efficiency of surfactant washing was assessed by the cytotoxicity of the matrix in the NIH 3T3 fibroblast culture. Viability and metabolic activity of cells were assessed via vital staining with a complex of fluorescent dyes LIVE/DEAD ® and PrestoBlue™ (Invitrogen, USA). Morphological examination of the liver cell-engineered constructs was carried out through histological staining and scanning electron microscopy with lanthanide contrast. Results. It was shown that the liver decellularization method used allows to obtain a biocompatible matrix with a residual DNA quantity <1%, which is capable of maintaining adhesion and proliferation of hAT-MSCs and HepG2. On day 7 of cultivation in the bioreactor, there was formation of a single conglomerate of the DHLF matrix with numerous groups of viable cells with a high nuclear-cytoplasmic ratio. The urea content in the culture medium is 1.5 ± 0.1 mmol/L, exceeding that of samples obtained under static conditions. This indicates the metabolic activity of HepG2 in the composition of the obtained culture systems. It was shown that constant flow of the culture medium in the perfusion bioreactor increased the proliferative activity of HepG2 and allowed to provide a more uniform colonization by matrix cells in comparison with static cultivation conditions. Conclusion. The conditions for uniform colonization of DHLFs in a flow-through bioreactor with cell cultures were established. The ability of the matrix to maintain adhesion and proliferation of hADSCs and HepG2 for 11 days indicates that it could be used in liver tissue engineering.