Evaluation of in vitro cytotoxicity of ACVR-IN-01 in a human mesenchymal stromal cell model

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%.

单纯疱疹病毒感染是一组由单纯疱疹病毒引起的疾病，其特征为皮肤、黏膜、中枢神经系统以及有时其他器官的损伤。病原体具有独特的生物学特性，这些特性影响了发病机制及随后的人类疾病。病毒具有穿透和复制于中枢神经系统，以及于背根神经节建立潜伏感染的能力。所有临床形式的疱疹感染均对抗病毒药物敏感。其中效力最显著者为佐维拉克斯（别名：阿昔洛韦，维罗莱克斯）。本研究的目的是通过测定培养的人间充质干细胞（体外）的总存活细胞数以及细胞培养的免疫表型研究来评估ACVR-IN-01的细胞毒性。研究结果表明，不同浓度的“ACVR-IN-01”对CD 90、CD 105、CD 34和CD 45抗原的表达产生了影响，并确定了“ACVR-IN-01”浓度增加与CD 73抗原表达减少之间的相关性。通过自动细胞计数器“Countess II”（赛默飞世尔科技，美国）检测培养间充质干细胞（MSCs）的功能状态，以评估其总存活细胞数。采用流式细胞仪进行细胞培养的免疫表型研究。在实验组和对照组的培养瓶中，从培养开始的第一、第二和第七天，使用针对CD34、CD45、CD73、CD90和CD105（贝克顿·迪金森和贝克曼库尔特，美国）的荧光标记抗体在FACSCanto II流式细胞仪（贝克顿·迪金森，美国）上确定细胞培养的免疫表型，操作遵循制造商的说明。为了确定培养中MSCs的存活率，我们使用了结合于富含鸟嘌呤和胞嘧啶区域DNA分子的核染料7-氨基乙酰胞嘧啶（7-AAD），细胞与7-AAD的结合表明细胞膜完整性的破坏。因此，具有荧光信号的细胞不包括在分析中。在实验培养开始7天后，在不同浓度的测试物质生长培养基中，评估了间充质干细胞上CD73的表达：图（a,b,c）- 25 μg/mL，图（d,e,f）- 200 μg/mL，图（g,h,i）- 400 μg/mL。注意：白色轮廓标记了直接（图a、d和g）和侧面（图b、e和h）的光散射，红色轮廓标记了碎片。以蓝色轮廓标记了抗体表达适中且活性降低20%和30%的区域（c和f）。橙色轮廓（i）表示抗体表达缺失，活性显著降低85%。