Abstract—

The use of mesenchymal stem cells (MSCs) with a pronounced immunomodulatory activity is a promising trend in the development of biomedical cell preparations (BMCPs). In oncohematology, the use of BMCPs containing MSCs is aimed at supporting hematopoiesis after cotransplantation with hematopoietic stem cells (HSC) and suppressing immune conflicts in allogeneic unrelated transplantation and severe autoimmune processes. An obligatory step in BMCP registration consists in confirmation of the MSCs’ authenticity, which includes the establishment of morphological characteristics, the assessment of the expression of specific markers and proteins, and confirmation of the cell genetic stability during cultivation. Determination of genetic-stability markers is possible using various methods; however, according to the recommendations of the American National Standardization Institute, the standard is the analysis of short tandem repeats (STR analysis). The purpose of this study is to develop the algorithm for determining the authenticity of BMCPs containing MSCs including STR analysis. MSCs were identified in BMCPs on the basis of the presence of immunological markers and spindle-cell adhesion to plastic. The number of viable cells was counted with a Goryaev’s chamber. Immunophenotypic characteristics of MSCs were assessed by flow cytometry. The level of production of specific proteins was assayed with enzyme immunoassay. Markers of genetic stability were identified by STR analysis. The methods were tested in triplicate for ten BMCP samples to confirm the reproducibility and reliability of the results. The algorithm developed for determining the BMCP authenticity has a high accuracy, as it includes the STR analysis technique, which makes it possible to identify 19 polymorphic STR markers located on different alleles. Using this method will allow BMCP manufacturers to pass the procedure of state registration of drugs.

中文翻译：

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一种确定含有间充质干细胞的生物医学细胞制剂真实性的算法

摘要-

使用具有显着免疫调节活性的间充质干细胞（MSC）是生物医学细胞制剂（BMCP）发展的一个有希望的趋势。在肿瘤血液学中，使用含有间充质干细胞的BMCP旨在支持与造血干细胞（HSC）共移植后的造血作用，并抑制同种异体无关移植和严重自身免疫过程中的免疫冲突。BMCP注册的必要步骤是确认MSC的真实性，包括建立形态特征、评估特定标记和蛋白质的表达以及确认培养过程中的细胞遗传稳定性。可以使用多种方法确定遗传稳定性标记；然而，根据美国国家标准化研究所的建议，标准是短串联重复序列的分析（STR分析）。本研究的目的是开发用于确定含有 MSC 的 BMCP 真实性的算法，包括 STR 分析。根据免疫标记物的存在以及梭形细胞与塑料的粘附，在 BMCP 中鉴定出 MSC。用 Goryaev 室计数活细胞的数量。通过流式细胞术评估 MSC 的免疫表型特征。用酶免疫测定法测定特定蛋白质的产生水平。通过 STR 分析鉴定遗传稳定性标记。该方法对 10 个 BMCP 样品进行了三次测试，以确认结果的再现性和可靠性。为确定 BMCP 真实性而开发的算法具有很高的准确性，因为它包含 STR 分析技术，可以识别位于不同等位基因上的 19 个多态性 STR 标记。使用这种方法将使BMCP生产商能够通过国家药品注册程序。