Engineering the Ovarian Follicle Microenvironment: Co-Culture Strategies and Medium Formulations for the Survival and Growth of Primary Ovarian Follicles Encapsulated in Alginate Hydrogels

Lifesaving cancer treatments, such as chemotherapy and radiation, threaten the fertility of young girls and women by diminishing the ovarian follicle (oocyte and surrounding cells) pool and triggering premature ovarian failure. To restore fertility, without the risk of reintroducing cancer cells post-treatment, in vitro follicle culture and fertilization techniques could be used to produce mature eggs, and subsequently embryos, from cryopreserved ovarian tissue. Remarkable progress has been achieved by engineering biomaterial scaffolds, such as alginate hydrogels, to maintain the three-dimensional architecture and cell-cell interactions of developing follicles. Hydrogel-encapsulating culture systems for ovarian follicles support the in vitro growth of secondary and multilayer follicles from a variety of species including mice, non-human primate, and human; however, the culture of early stage (primordial and primary) follicles, which are more abundant in the ovary and survive cryopreservation, has been challenging. Early stage follicles have been grown via in situ organ culture or two-step culture systems, in which ovarian tissue fragments containing early stage follicles are cultured in vitro.;Inspired by ovarian organ culture, we developed co-culture strategies for the in vitro culture of isolated early stage follicles. Biomaterials (alginate hydrogels) were employed to support the three-dimensional structure of developing follicles. Co-culture with ovarian stromal cells (theca-interstitial cells or TICs) and mouse embryonic fibroblasts (MEFs) enabled the survival and growth of early secondary (90-100 mum) and primary mouse follicles (60-80 mum) capable of producing metaphase II (MII) eggs. Paracrine signaling between the feeder cells and follicle was a major contributor to follicle survival and growth. While effective, these co-culture methods were undefined. Thus, based on embryonic stem cell culture medium that replaced MEFs, we designed novel feeder-free medium formulations and identified critical factors in primary follicle development. The maximal medium formulation was identified as alphaMEM/F12 supplemented with fetuin, insulin, transferrin, selenium, and follicle stimulating hormone. Moreover, ascorbic acid and lower oxygen tension were discovered to significantly improve follicle survival. Taken together, these advancements are enabling tools for future basic science discoveries and fertility preservation technologies.