Objective. Evaluate the cytotoxicity of a new chemical compound «ACVR-IN-01» (in vitro) on a culture of human mesenchymal stromal cells (MSCs). Materials and methods. The characterized MSC culture of the 4th passage from the biobank of cell cultures was used. Test samples «ACVR-IN-01» were prepared at concentrations of 25, 200 and 400 μg/ml. For each tested concentration of «ACVRIN-01» cultivation was carried out in triplicate. A visual assessment of the state of the cell culture was performed after 24, 48 hours and 7 days. The assessment of the functional state of MSCs was carried out by identifying the total number of viable cells and immunophenotypic studies of cell culture. Results. Cultivation of MSCs in a complete growth medium with «ACVR-IN-01» at a concentration of 25, 200 and 400 μg/ml for 7 days does not affect the expression of CD90 and CD105 antigens at all points of observation in vitro. Cultivation of MSCs in a complete growth medium with «ACVR-IN-01» at concentrations of 25, 200 and 400 μg/ml for 7 days did not cause the expression of CD34 and CD45 antigens at all in vitro observation points. Conclusion. The chemical compound «ACVR-IN-01» does not have a significant effect on the linear affiliation of MSCs. Evaluation of the cytotoxicity of the test substance «ACVR-IN-01» (400 μg/ml) on a cell culture in a complete growth medium showed a pronounced decrease in the expression of the CD73 antigen on the 7th day of the experimental in vitro study, accompanied by a loss of the differentiation potential of MSCs in the osteogenic and chondrogenic direction. Key words: herpes virus infection, cytotoxicity, mesenchymal stromal cells, «ACVR-IN-01»
Purpose: To evaluate the influence of a humic-fulvic acid substance on the quantitative yield of residual foci of the DNA double-strand break (DSB) repair protein-marker - phosphorylated histone H2AX (γH2AX) and proliferation activity in a culture of human mesenchymal stromal cells (MSCs) 24, 48, and 72 h after exposure to X-ray radiation at doses of 2, 4 and 10 Gy. Material and methods: Through 24 hours after incubation of MSCs with a substance of humic-fulvic acids (Humic Complex, OOO Sistema-BioTechnologies, Russia) at a dilution of 1/1000. Cells were irradiated on an X-ray biological device RUB RUST-M1 at a voltage of 200 kV, beam current 2×5 mA, aluminum filter 1.5 mm, absorbed dose rate 0.85 Gy/min. Immunocytochemical staining was used to quantify the residual γH2AX foci and the percentage of proliferating cells using antibodies to γH2AX and Ki-67 (a marker protein for cell proliferation), respectively. Statistical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). To assess the significance of differences between samples, Student’s t-test was used. Results and conclusion: The conducted studies showed that on the cell model used and under the above experimental conditions, the humic-fulvic acid substance does not affect the efficiency of repair of radiation-induced DNA DSBs, however, it significantly reduces the proliferation activity of both irradiated and non-irradiated MSCs. It is advisable to conduct detailed studies of the molecular and cellular mechanisms of the antiproliferative effect of humic and fulvic acids.
Purpose: To evaluate the frequency and spectrum of chromosome aberrations under X-Ray exposure at doses of 80, 250, and 1000 mGy in a human multipotent mesenchymal stromal cell (MMSC) cell line during long-term cultivation. Material and methods: MMSCs were isolated from human gingival mucosa by an enzymatic method and cultured in a serum-free medium. The presence of surface antigens was determined using the method of flow cytometry. The ability of the cell line to differentiate in the osteogenic, adipogenic, and chondrogenic directions was studied using induction media. Authentication was performed by genotyping of polymorphic STR loci, cytogenetic analysis was performed by multicolor fluorescent in situ hybridization (mFISH). Irradiation was carried out on an X-ray biological unit RUB RUST-M1 (Russia) at a dose rate of 40 mGy/min, a voltage of 100 kV, and a current of 0.8 mA. Results: At the first passage after irradiation, a statistically significant increase in the frequency of non-clonal CA compared with the control was recorded at a dose of 80, but not 250 and 1000 mGy. At the late stages of cultivation, the average frequency of breaks per chromosome in the group of non-irradiated cells did not differ from the values obtained after irradiation at doses of 80, 250, and 1000 mGy (p > 0.05). However, in MMSCs irradiated at a dose of 80 mGy, damage occurred more often in pairs of chromosomes 6 and 10, and at a dose of 1000 mGy, in a pair of chromosomes 9. A single irradiation of MMSCs in vitro did not affect the growth and progression of MMSCs characteristic of the studied primary cell line, of clonal cells with chromosome translocations and monosomy X, but led to an increase in the representation of a clone with tetrasomy 8. The total number of random clones with chromosome translocations that arose de novo increased after irradiation at a dose of 1000 mGy. Conclusion: Minor fluctuations in the proportion of cells with non-clonal CA, depending on the dose received in the early stages after irradiation (passage 1–4), disappeared at the later stages of cultivation (passage 8–14). There were no differences in mean frequencies between irradiated and non-irradiated MMSCs, but after irradiation, damage to some chromosomes could occur more frequently than others. A single X-ray irradiation of MMSCs can promote the growth and progression of primary pathological cytogenetic clones, regardless of the dose received, as well as an increase in the total number of de novo cell clones with chromosomal translocations that have arisen. A single X-ray irradiation of MMSCs can promote the growth and progression of primary pathological cytogenetic clones, regardless of the dose received, as well as an increase in the total number of de novo cell clones with chromosomal translocations that have arisen.
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