Protocol for generating and culturing high-grade serous ovarian carcinoma organoids from fresh or cryopreserved patient samples

Stable long-term high-grade serous ovarian carcinoma (HGSC) organoids can be difficult to develop due to limited primary sample growth in 3D conditions. Here, we present a detailed protocol for the development of stable HGSC organoids, including all steps from seeding primary cells through culture monitoring, passaging, and expansion to viable cryopreservation and resuscitation. We provide guidance on initial passaging, passaging ratios, and criteria for successful culture establishment. Moreover, we include a troubleshooting section tackling recurrent problems in HGSC organoid development. For complete details on the use and execution of this protocol, please refer to Senkowski et al . 1