We have refined the technique for isolating and propagating cultures of primary epithelial ovarian cancer (EOC) cells derived from solid tumors and ascites. Both protocols involve a simple yet rapid method for the growth and propagation of EOC tumor and ascites cells in a basal culture medium without the addition of growth factors. Isolation of tumor EOC cells involves the mechanical disruption of the tumor tissue with the help of a cell scraper, while ascites-derived EOC cells are mixed with growth medium and placed directly into culture with very little manipulation. We further describe a partial trypsinization method to eliminate fibroblast contamination from primary EOC cells derived from solid tumors. These methods allow for the direct application of many molecular, cellular, and functional analyses within a few weeks of initial isolation, with the added potential of retrospective analyses of archived cells and tissues. Thus, we have included steps for long-term cryopreservation of early-passage EOC cells. Initial isolation of EOC cells can be completed within 1 h, and primary cells are further expanded in culture for several weeks.
