(594g) Primary Ovarian Follicle Development Is Promoted within Alginate Hydrogels Via Co-Culture with Theca-Interstitial Cells and Mouse Embryonic Fibroblasts

In vitro culture systems for ovarian follicles (oocyte and supporting cells) are enabling tools for the study of folliculogenesis and the advancement of fertility preservation technology for cancer survivors (oncofertility). Three-dimensional follicle culture systems have been engineered using hydrogels, which maintain the spherical morphology of the follicle. To date, these culture systems have achieved the consistent growth of mouse secondary follicles, which have yielded healthy offspring; however, further advancements are necessary in order to promote the development of early stage (primordial and primary) follicles. Ovarian stromal cells (connective tissue of the ovary) have significant roles in early follicle development; nevertheless, the current state-of-the-art follicle culture systems do not utilize these cells. Hence, we hypothesize that the co-culture of stromal or theca-interstitial cells (TICs) will promote the development of early stage mouse follicles. Our co-culture system is built directly upon the existing alginate hydrogel culture system, which supports secondary follicles as small as 120 µm. Follicles are encapsulated within 0.25% alginate hydrogel beads and cultured for 14 days within separate wells of a 96-well culture plate. The TICs are seeded on the bottom of the well, directly beneath the floating hydrogel. Early-secondary (100-110 µm) and late-primary follicles (90 µm) survived, increased in size to 300 µm, developed antral cavities, and produced mature (MII) eggs. The MII rate from antral follicles was approximately 50%. Without co-culture, the follicles do not survive. To determine if this growth stimulation was specific to TICs, we tested mouse embryonic fibroblasts (MEFs) (feeder cells for stem cell culture). MEF co-culture promoted the development of smaller primary follicles (70-80 µm) and production of MII eggs. Therefore, we have demonstrated that TIC/MEF co-culture promotes the development of primary follicles as small as 70 µm in the alginate hydrogel culture system. Our next steps include elucidating the underlying mechanisms by testing additional cell types and identifying the critical paracrine factors.