Co-culturing matrigel-encapsulated post-cryopreserved human ovarian cortical tissue with mesenchymal stem cells to improve In situ follicular activation and early development.

Recent advances in culturing cryopreserved ovarian cortical tissue (OCT) offer a promising strategy for fertility preservation, especially for patients undergoing gonadotoxic treatments. This study evaluated whether Matrigel encapsulation combined with mesenchymal stem cell (MSC) co-culture enhances folliculogenesis in post-cryopreserved human OCT during an 8-day in vitro culture. Thawed human OCT fragments were encapsulated in Matrigel and cultured in the upper chamber of a transwell plate, with MSCs in the lower chamber. Follicular survival and maturation were evaluated histologically, apoptosis by TUNEL, and proliferation by Ki67 expression. QRT-PCR quantified transcripts of FIGLA, GDF9, and FSHR, and endocrine function was measured through 17-β estradiol (E2) levels using ELISA. Compared with the control and Matrigel-only (Mat) groups, the MSC-Mat combination significantly increased the transition of primordial follicles to secondary follicles on days 4 and 8, while the percentage of normal follicles remained similar across groups. The MSC-Mat group showed fewer apoptotic cells on days 2 and 8 and higher proliferative activity on days 2, 4, and 8. Gene analysis confirmed enhanced follicular development in the MSC-Mat group, demonstrated by a progressive rise in FSHR expression, peaking on day 8. Both Mat and MSC-Mat groups showed increasing E2 secretion over time, with the highest levels in the MSC-Mat group by day 8. Overall, Matrigel encapsulation combined with MSC co-culture did not affect follicle survival but markedly promoted follicular activation and early development in post-cryopreserved human OCT, supporting its potential clinical use in fertility preservation.