Ex-Vivo and In-Vivo Expansion of Spermatogonial Stem Cells Using Cell-Seeded Microfluidic Testis Scaffolds and Animal Model.

Aims: This study was designed to provide both ex-vivo and in-vivo methods for the extraction and expansion of spermatogonial stem cells (SSCs).

Methods: For in-vivo experiments, azoospermic mouse model was performed with Busulfan. Isolation, culture, and characterization of neonate mouse SSC were also achieved. We performed an in-vivo injection of labeled SSCs to the testes with azoospermia. In ex-vivo experiments, extracted SSCs were seeded on the fabricated scaffold consisting of hyaluronic acid (HA) and decellularized testis tissues (DTT). Immunofluorescence staining with PLZF, TP1, and Tekt 1 was performed for SSCs differentiation and proliferation.

Results: Several studies demonstrated efficient spermatogenic arrest in seminiferous tubules and proved the absence of spermatogenesis. Transplanted SSCs moved and settled in the basement covering the seminiferous tubules. Most of the cells were positive for Dil, after 4 weeks. An epithelium containing spermatogonia-like cells with Sertoli-like, and Leydig cells were evident in the seminiferous tubules of biopsies, and the IHC staining was significantly positive, 4 weeks after injection of SSCs. The results of the ex-vivo experiments showed positive staining for all markers, which was significantly enhanced in scaffolds of ex-vivo experiments compared with in-vitro seeded scaffolds.

Conclusion: Ex-vivo SSC differentiation and proliferation using cell-seeded microfluidic testis scaffolds maybe effective for treatment of the azoospermia.