Self‐Organizing Ovarian Somatic Organoids Preserve Cellular Heterogeneity and Reveal Cellular Contributions to Ovarian Aging

Ovarian somatic cells are essential for reproductive function, but no existing ex vivo models recapitulate the cellular heterogeneity or interactions within this compartment. We engineered an ovarian somatic organoid model by culturing a stroma-enriched fraction of mouse ovaries in scaffold-free agarose micromolds. Self-organized ovarian somatic organoids maintained diverse cell populations, produced extracellular matrix, and secreted hormones. Organoids generated from reproductively old mice exhibited reduced aggregation and growth compared to young counterparts, as well as differences in cellular composition. Interestingly, matrix fibroblasts from old mice demonstrated upregulation of pathways associated with the actin cytoskeleton and downregulation of cell adhesion pathways, indicative of increased cellular stiffness that may impair organoid aggregation. Cellular morphology, which is regulated by the cytoskeleton, significantly changed with age and in response to actin modulation. Moreover, actin modulation altered organoid aggregation efficiency. Overall, ovarian somatic organoids have advanced knowledge of cellular contributions to ovarian aging.