Dynamic Culture System Supports Mouse Spermatogenesis in Cell Culture Medium

Introduction: Decellularizing testis tissue and recellularizing with spermatogonial stem cells (SSCs) seems to be a promising approach to restore fertility in prepubertal boys who undergoes cytotoxic therapies. Method: Testis tissue decellularization was performed by adding ۱% SDS and confirmed by histological analysis and DNA quantification. The MTT assay was performed for biocompatibility analyses. SSCs were derived from male mice and cultured in αMEM medium for two weeks. Expanded SSCs were seeded onto the DTM scaffold. The recellularized DTM scaffold disc was cultured in a static cultivation system for one week, then transferred in a dynamic mini-perfusion bioreactor for two weeks. The expression of Id۴, Plzf, Gfrα, Prm, Sycp۳, ABP, Ki۶۷, Bax, and Bcl۲ genes were assessed in SSCs and recellularized DTM after static and dynamic cultivations. Result: DNA qualification indicated that approximately ۹۹% of the DNA components were removed from DTMs. Hematoxylin-eosin, Masson's trichrome, and DAPI staining confirmed the effective recellularization. Dynamic cultivation of recellularized DTMs at the flow rate of ۱۰ ml/h provided optimum conditions. The expression of SSCs-specific genes of Id۴, Plzf, and Gfrα-۱ and post-meiosis genes of Scp۳, prm۱, and ABP was insignificantly higher in the DTMs group than in the control group. Ki۶۷ expression was shown no difference between groups. An insignificant lower expression of the Bax and higher expression of Bcl۲ genes was detected in the DTMs group compared to the control. Conclusion: Our results indicated that SSCs could successfully be attached to the DTMs and effectively proliferate in the mini-perfusion bioreactor.

کلیدواژه ها:

Spermatogonial stem cell ، Decellularized testicular matrix ، Mini-perfusion bioreactor