Washington University in St. Louis.
Washington University in St. Louis; University of California San Francisco.
J Vis Exp. 2023 Jan 6(191). doi: 10.3791/64878.
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.
类器官是 3D 动态肿瘤模型，可成功从患者来源的卵巢肿瘤组织、腹水或胸腔积液中生长，并有助于发现新的治疗方法和卵巢癌预测性生物标志物。这些模型再现了克隆异质性、肿瘤微环境以及细胞-细胞和细胞-基质相互作用。此外，它们在形态、细胞学、免疫组织化学和遗传学上与原发性肿瘤相匹配。因此，类器官促进了对肿瘤细胞和肿瘤微环境的研究，优于细胞系。本方案描述了从患者肿瘤、腹水和胸腔积液样本中生成患者来源的卵巢癌类器官的独特方法，成功率高于 97%。患者样本通过机械和酶消化分离成细胞悬浮液。然后，将细胞铺板于基底膜提取物 (BME) 上，并使用含有针对高级别浆液性卵巢癌 (HGSOC) 培养的特定补充剂的优化生长培养基进行支持。形成初始类器官后，PDO 可以进行长期培养，包括传代以进行后续实验的扩增。
