Obtaining rat testicular cell cultures for research and development of biological drugs for spermatogenesis restoration

Aim. To create rat testicular cell collections characterized by pheno­types and functional activity to determine optimal conditions for their use in models for assessing the specific activity of spermatogenesis restoration drugs.

Material and methods. To isolate different cell types from rat tes­tes, sequential enzymatic treatment of testicular tissue was used. Immu­no­cytochemical analysis of the isolated cell cultures was performed for canonical and non-canonical markers. Testosterone secretion by Leydig cells was measured on days 2, 4, and 7 of culture using enzyme-­linked immunosorbent assay. The developed model for assessing the biological activity of drugs in Leydig cells was validated in accordance with the requirements of the Eurasian Economic Commission Decision dated July 17, 2018, № 113.

Results. The following cell cultures were isolated from rat testes and characterized: Leydig cells expressing specific CYP11A1 and LHR markers; resident mesenchymal stromal/stem cells expressing CD73, CD90, and PDGFRb markers; Sertoli cells expressing inhibin beta B and Sox9 markers; spermatogonial stem cells expressing GDNFR, c-kit, and alkaline phosphatase markers; and peritubular myoid cells expressing αSMA. It was shown that Leydig cells, when isolated in culture, began ex­pressing vinculin, and with prolonged cultivation and passaging, they acquired the Sox9 and vimentin markers. In addition, Leydig cells in culture sharply reduced their ability to secrete testosterone within a week. Based on these data, our previously developed in vitro mo­del for assessing the specific activity of drugs for spermatogenesis resto­ration was optimized and validated.

Conclusion. The approaches used enable the formation of charac­te­rized collections of different cell types isolated from the testes of la­bo­ratory animals. However, when isolated from tissue, due to the loss of a specific microenvironment, the phenotypic and functional properties of the cells may change over time in culture. This should be taken into account when creating and subsequently using cell collections. A study was conducted on the phenotype of testicular cells, as well as the secretory activity of Leydig cells. Based on this, a method for asses­sing specific activity in vitro and an optimal biobanking strategy were de­veloped.

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