Probing the Ovarian Microenvironment: Methods for Isolation, 2D Culture, and Organoid Culture of Mouse Ovarian...

The ovary consists of heterogeneous populations of somatic cells, both within the follicle and the surrounding stroma, which are critical to support ovarian function and for the generation of high-quality gametes. We report methods for isolating somatic cells from mouse ovaries, including endothelial, epithelial, steroidogenic, stromal, and immune cells. When these primary ovarian somatic cells are plated and cultured in a traditional 2D culture system, the cellular heterogeneity, organization, as well as cell-cell and cell-matrix interactions typically found in the ovary are lost. Thus, we also describe how to generate mouse ovarian somatic organoids using a scaffold-free approach. These organoids self-assemble, maintain diverse cell populations, and produce extracellular matrix and secreted factors, including cytokines. Organoids can be utilized for co-culture experiments and can be maintained in culture for at least 3 weeks with high viability. Overall, these models enable interrogation of ovarian physiology and pathology from the somatic cell perspective.