An empirically refined workflow for generating patient-derived organoids from epithelial ovarian cancer

Patient-derived organoids (PDOs) can recapitulate selected features of original tumors and provide a useful system for studying epithelial ovarian cancer (EOC) in three-dimensional culture. Nonetheless, the protocol to generate EOC-PDOs has not yet been standardized. We therefore empirically refined the culture conditions and medium composition, considering the protocol effective when EOC PDOs were successfully established in five consecutive attempts. Tissue and ascites samples (from 29 and 8 patients, respectively) were used and the Syringe-extruded Organoid Basement membrane extract Assembly (SOBA) fragment culture method applied. Selected similarities in cell morphology, marker expression, and detectable alterations between organoids and patient-matched tumor tissue were assessed using pairwise comparisons. The refined workflow used a culture medium containing recombinant human R-Spondin 1 protein (250 ng/mL), Noggin, and NRG1, factors implicated in organoid maintenance and expansion. Ultimately, 31 EOC organoid lines successfully generated from 82.8% (24/29) of tissue and 87.5% (7/8) of ascites samples. The SOBA technique increased the 10 day PDO yield by 2.75-fold relative to the surface-attached dome method (p < 0.0001). This empirically refined workflow can be adjusted for specific experimental applications; however, further validation across larger and clinically diverse EOC cohorts is required.