Cryopreserved fragments of testicular seminiferous tubules of rats as a source of spermatogonial stem cells

Abstract

The use of modern technologies of cryopreservation of testicular tissue samples in prepubertal patients is one of the ways to maintain their fertility in the future. The purpose of the study was to investigate the proliferative potential, morphological characteristics and expression of specific markers of cell culture obtained from cryopreserved and vitrified fragments of seminiferous tubules (FSTs) of rats' testis. Materials and methods. The isolation of cells from native, cryopreserved and vitrified FSTs of immature rats was performed by incubation in a solution of collagenase type IV (1 mg/mL) + DNase (500 μg/mL). Cell viability was determined by Trypan blue staining. Monoclonal antibodies CD9-FITC, CD24-PE, CD45-FITC, CD90-FITC were used for immunophenotype analysis. Morphological characteristics, proliferative activity (MTT assay), relative number of cells positive for MAGE-B1 and vimentin were assessed in the obtained cultures. Results. The analysis of phenotypic characteristics showed that cells from native, cryopreserved and vitrified FSTs were characterized by high expression level of CD9 (≥ 40 %), CD24 (≥ 70 %), CD90 (≥ 70 %) and low expression of the CD45 (≤ 1 %). In cell culture in vitro, the studied cells from cryopreserved and vitrified rat's FSTs had the ability to adhere and proliferate while maintaining a cells population positive for MAGE-B1 and vimentin. Conclusions. The results can be the basis for the development of effective protocols for the cultivation and cryopreservation of testicular spermatogonial stem cells in order to restore fertility in men.