Evaluation of in vitro cytotoxicity of ACVR-IN-01 in a human mesenchymal stromal cell model
Description
Herpes infection is a group of diseases caused by the herpes simplex virus, which are characterized by damage to the skin, mucous membranes, central nervous system, and sometimes other organs. Pathogens have unique biological properties that affect the pathogenesis and subsequent human disease. Viruses have the ability to penetrate and replicate in the central nervous system, as well as the ability to establish latent infection in the dorsal root ganglia. Herpetic infection in all clinical forms is susceptible to the effects of antiviral drugs. The most potent of these is Zoviraxum (synonyms: Aciclovir, Virolex). The purpose of this work was to assess the cytotoxic properties of ACVR-IN-01 on the culture of human mesenchymal stromal cells (in vitro) by determining the total number of viable cells and immunophenotypic studies of the cell culture. As a result of the work, the effect of various concentrations of "ACVR-IN-01" on the expression of CD 90, CD 105, CD 34 and CD 45 antigens was assessed, a correlation was determined between an increase in the concentration of "ACVR-IN-01" and a decrease in the expression of CD 73 antigen. The functional state of mesenchymal stromal cells (MSCs) was assessed by detecting the total number of viable cells using an automatic cell counter, “Countess II” (Thermo Fisher Scientific, USA). An immunophenotypic study of the culture was carried out using a flow cytometer. The immunophenotype of the cell culture was determined on the first, second, and seventh days from the start of cultivation in the experimental and control flasks. The expression of surface markers was assessed using fluorochrome-labelled antibodies against CD34, CD45, CD73, CD90, and CD105 (BD Biosciences and Beckman Coulter, USA) on a FACSCanto II flow cytometer (Becton Dickinson CA, USA) according to the manufacturer's instructions. To determine the viability of MSCs in culture, we used the nuclear dye 7-aminoactinomycin D (7-AAD), which binds to DNA molecules at the regions that are rich in guanine and cytosine. Cell binding to 7-AAD indicated a violation of membrane integrity. Therefore, cells with fluorescent signals were not included in the analysis. Expression of CD73 on mesenchymal stem cells 7 days after the start of experimental cultivation in a growth medium with different concentrations of the test substance: Figures (a,b,c) - 25 μg/mL, (d, e, f) - 200 μg/mL, (g, h, i)-400 μg/mL. Note: the white contour marks the direct (Figures a, d and g) and lateral (Figures b, e and h) light scattering, the red contour marks the debris. Areas of moderate antibody expression with a decrease in activity by 20% and 30%, respectively, are marked with a blue outline (c and f). The orange outline (i) denotes the absence of antibody expression, with a pronounced decrease in activity by 85%.
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Cultivation: One day prior to the experiment, MSCs were seeded in culture flasks with an area of 25 cm2 at a concentration of 20 × 103 cells per cm2 of the bottom surface of the flask and cultured in a complete growth medium without xenogeneic components (xeno-free) (Mesencult, Stem Cell, USA) with the addition of 100 U/ml penicillin, 100 U/ml streptomycin, and 2 ml of glutamine in a CO2 incubator at 37 °C, 5 % CO2, and 80 % humidity. For each investigated concentration of ACVR-IN-01, cultivation was carried out in triplicate. MSCs from the same sample and passage were used as controls. After 24 h of cultivation, all the cell cultures were visually assessed, the growth medium was selected, and all the flasks were washed with Dulbecco's buffer solution (Biological Industries, USA). Afterwards, 5 mL of the growth medium containing the investigated concentration of the drug was introduced into the experimental vials. In the control vials, the growth medium, which lacked the study drug, was provided in volumes identical to those of the experimental groups. After 24 h, 48 h, and 7 days of cultivation, a visual assessment was carried out in all the test flasks to determine the functional state of the cells at the indicated observation points. One culture flask was taken from each experimental and control sample, from which the medium was removed by washing the flasks with Dulbecco's buffer solution (Biological Industries, USA). Next, a suspension of cells with 0.25 % trypsin-EDTA solution (Stem Cell, USA) was centrifuged at 1200 rpm for 7 min. The cell pellet was resuspended in complete growth medium. The functional state of mesenchymal stromal cells: The functional state of MSCs was assessed by detecting the total number of viable cells using an automatic cell counter, “Countess II” (Thermo Fisher Scientific, USA). An immunophenotypic study of the culture was carried out using a flow cytometer. The immunophenotype of the cell culture was determined on the first, second, and seventh days from the start of cultivation in the experimental and control flasks. The expression of surface markers was assessed using fluorochrome-labelled antibodies against CD34, CD45, CD73, CD90, and CD105 (BD Biosciences and Beckman Coulter, USA) on a FACSCanto II flow cytometer (Becton Dickinson CA, USA) according to the manufacturer's instructions. To determine the viability of MSCs in culture, we used the nuclear dye 7-aminoactinomycin D (7-AAD), which binds to DNA molecules at the regions that are rich in guanine and cytosine. Cell binding to 7-AAD indicated a violation of membrane integrity. Therefore, cells with fluorescent signals were not included in the analysis.