Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR-T cell testing

Glioblastoma tumors exhibit extensive inter- and intra-tumoral heterogeneity, which has contributed to poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Current in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include methods for passaging and cryopreserving GBOs for continued use, biobanking, and long-term recovery. We further describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. This protocol takes approximately 2-4 weeks to generate GBOs and 5-7 days to perform CAR-T cell co-culture. Competence with human cell culture, tissue processing, immunohistology, and microscopy is required for optimal results. Short Summary Detailed procedures for generating and biobanking glioblastoma organoids from resected patient tumor tissue and testing CAR-T cell efficacy by co-culture. Additional procedures for tissue processing, immunohistology, and detecting hypoxia gradients and actively proliferating cells. Associated Link Box for Key Reference Jacob, F. et al. A Patient-derived glioblastoma organoid model and biobank recapitulates inter- and intra-tumoral heterogeneity. Cell 180, 188-204 e122, doi:10.1016/j.cell.2019.11.036 (2020).