Abstract
Ex vivo follicle growth is an essential tool, enabling interrogation of folliculogenesis, ovulation, and luteinization. Though significant advancements have been made, existing follicle culture strategies can be technically challenging and laborious. In this study, we advanced the field through development of a custom agarose micromold, which enables scaffold-free follicle culture. We established an accessible and economical manufacturing method using 3D printing and silicone molding that generates biocompatible hydrogel molds without the risk of cytotoxicity from leachates. Each mold supports simultaneous culture of multiple multilayer secondary follicles in a single focal plane, allowing for constant timelapse monitoring and automated analysis. Mouse follicles cultured using this novel system exhibit significantly improved growth and ovulation outcomes with comparable survival, oocyte maturation, and hormone production profiles as established three-dimensional encapsulated in vitro follicle growth (eIVFG) systems. Additionally, follicles recapitulated aspects of in vivo ovulation physiology with respect to their architecture and spatial polarization, which has not been observed in eIVFG systems. This system offers simplicity, scalability, integration with morphokinetic analyses of follicle growth and ovulation, and compatibility with existing microphysiological platforms. This culture strategy has implications for fundamental follicle biology, fertility preservation strategies, reproductive toxicology, and contraceptive drug discovery.