Protocol for generating an in vitro 3D multicellular culture model of ovarian high-grade serous carcinoma

The development of translational ovarian cancer models to investigate and overcome treatment resistance, while accounting for the impact of the tumor microenvironment, is critical. Here, we present a protocol to establish a multicellular culture model that retains both genetic complexity and the microenvironment of patient tumors and is amenable to molecular and phenotypic analyses and high-throughput drug testing. We describe steps for culturing and characterizing stromal cells derived from cryopreserved and fresh samples and detail procedures for combining them with organoids. • Instructions for obtaining patient-derived fibroblasts, mesothelial cells, and adipocytes • Steps for combining patient-derived organoids with tumor microenvironment components • Procedures for high-throughput drug sensitivity testing and single-cell RNA sequencing • Guidance on immunofluorescence staining and on generating mCherry-labeled organoids Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The development of translational ovarian cancer models to investigate and overcome treatment resistance, while accounting for the impact of the tumor microenvironment, is critical. Here, we present a protocol to establish a multicellular culture model that retains both genetic complexity and the microenvironment of patient tumors and is amenable to molecular and phenotypic analyses and high-throughput drug testing. We describe steps for culturing and characterizing stromal cells derived from cryopreserved and fresh samples and detail procedures for combining them with organoids.