Establishment and characterization of a 3D model culture system of primary human ovarian cells: Silk-Ovarioids

In vitro models that mimic ovaries are instrumental in unraveling physio-pathological mechanisms underlining follicle activation and growth. Three-dimensional (3D) systems that replicate the heterogeneity and cell-cell communication among different ovarian cell types are biologically most relevant. However, such models using human primary ovarian cells have not yet been established and standardized. Here, we developed and characterized long-term cultured 3D models of primary ovarian somatic cells, isolated from normal adult tissues, using Biosilk as a scaffold. We successfully created both cortex- and medulla-derived 3D systems (Silk-Ovarioids). Through transcriptomics, proteomics, and immunostaining, we identified the presence of key ovarian somatic cell types (granulosa, stroma, endothelial and perivascular cells). Notably, the Silk-Ovarioids exhibit the formation of a proangiogenic hypoxic core, as evidenced by the formation of vessel-like structures after 6 weeks of culture. The Silk-Ovarioids have low inter/intra-batch variability and long-term culture stability, highlighting their potential as a robust step towards a bioengineered patient-specific artificial ovary.