Reconstruction of ovarian follicular-like structure by recellularization of a cell-free human ovarian scaffold with mouse fetal ovarian cells.

The present study assessed the supportive roles of the decellularized human ovarian tissue in homing of mouse fetal ovarian cells into the scaffold as well as the formation of the follicular-like structure. The human ovarian cortical tissues were decellularized by three freeze-thaw cycles and then, treated with Triton X-100 for 15 h and 0.5% sodium dodecyl sulfate for 72 h. After isolation and preparation of mouse fetal ovarian cells (19 dpc) they were seeded into the decellularized scaffolds and cultured for 7 days, then using a light microscope, laser confocal scanning microscope, and scanning electron microscope these scaffolds were studied. Analysis of gene expression related to oocyte and follicular cells such as Ddx4, Nobox, Gdf9, and Connexin37 was assessed by real-time RT-PCR and the DDX4 and GDF9 proteins were detected by immunohistochemistry. The result showed that the human ovarian tissue was decellularized properly and the tissue elements and integrity were well preserved. After 7 days of in vitro culture, the fetal ovarian cells attached and penetrated into different sites and depths of the scaffold. The formed organoid within the scaffold showed large round, small polyhedral, and elongated spindle cells similar to the follicle structure. The molecular analysis and immunohistochemistry were confirmed an increase in the expression of genes and proteins related to oocyte and follicular cells in these reconstructed structures. In conclusion, the recellularization of human ovarian scaffolds by mouse fetal ovarian cells could support the follicular-like structure formation and it provides an in vitro model for follicle reconstitution and offers an alternative approach for clinical usage.