Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Abstract

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Figure 1:. Plating of patient-derived ovarian cancer organoids.

Representative image of the organoid plating. Aliquots of the organoid mixture are carefully plated, ensuring no bubbles are formed.

Figure 2:. Tumor tissue prior to dissection.

Representative image of tumor tissue obtained for organoid generation.

Figure 3:. Schematic of patient collections formed into organoids.

Patient tissue, ascites, or pleural fluid are mechanically or enzymatically digested. The single-cell suspension is then plated into BME, where the culture proliferates with the help of specialized growth media tailored to HGSOC.

Figure 4:. Representative images of two unique patient-derived ovarian cancer organoids after 7 days of growth.

The organoid images were taken at 40x. The brightfield scale bar on the left image is 100 μm and on the right image is 200 μm.

Figure 5:. Hematoxylin and eosin staining of PDO.

Representative image of H&E staining of an ovarian cancer organoid generated from a patient sample. The image was taken at 40x, and the scale bar is 50 μm.