Morphofunctional characteristics of spermatogonial stem and accessory-regulatory cells from cryopreserved fragments of testis

Abstract

The problem of preserving and restoring human reproductive potential currently remains one of the most acute medico-social issues worldwide. Low-temperature storage of testicular fragments from prepubertal patients offers an opportunity to restore their fertility in the future. Objective. The aim of this study was to determine the optimal method for isolating spermatogonial stem and accessory regulatory cells (SS and ARCs) from cryopreserved fragments of seminiferous tubules of testes (FSTT) and to investigate their morphofunctional characteristics. Materials and Methods. Isolation of SS and ARCs from the testicular fragments of immature rats was performed via homogenization and incubation in either a 0.25% trypsin solution or a combination of type IV collagenase (1 mg/mL) + DNase (500 μg/mL). The efficiency of FSTT disaggregation was determined by evaluating cell yield, viability (trypan blue exclusion test), and total metabolic activity (MTT assay). The obtained SS and ARCs from cryopreserved and vitrified FSTT were characterized by preservation of morphological characteristics in relation to native samples. Under cultivation conditions, isolated cells from all experimental groups showed the ability to proliferate with preservation of the CDH1-positive population. Results. It has been established that the disaggregation of immature rat FSTT is most effective when using a combination of type IV collagenase and DNase, based on the obtained results of cell yield, viability, and total metabolic activity. The SS and ARCs obtained from native, cryopreserved, and vitrified FSTTs were characterized by the preservation of morphological characteristics in relation to native samples. Under culture conditions, isolated cells from all samples demonstrated the capacity for proliferation while maintaining the CDH1-positive population. Conclusions. These data can serve as a foundation for developing effective methods for cultivating and freezing spermatogonial stem cells, which will contribute to the design of new therapeutic approaches for male infertility