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

Abstract

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. AbstractGlioblastoma tumors exhibit extensive inter-and intra-tumoral heterogeneity, which has contributed to the 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, and immunohistology is required for optimal results.An ideal in vitro model for glioblastoma would maintain cellular diversity, preserve native cell-cell interactions, recapitulate defining histological characteristics, and maintain the transcriptomes and mutation profiles of parent tumors. To be useful for personalized medicine approaches, the model would need a quick generation time with high fidelity for success and be scalable to test potential therapeutics. This would require maintaining patient-derived glioblastoma tissue in culture with minimal perturbation. Towards this goal, we opted to directly culture micro-dissected tumor pieces without cell dissociation to preserve cell-cell interactions and minimize clonal selection. We start from resected patient tumor tissue that can be sampled from different tumor foci or subdivided into regional samples to analyze intra-tumoral heterogeneity. Through testing of many medium formulations, we learned that tumor pieces did not require basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), serum, or added extracellular matrix such as Matrigel to survive, form organoids, and proliferate at rates similar to parent tumors 11 . We optimized a chemically defined medium that contains basic nutrients and components needed to support glial cell health with few exogenous factors to minimize clonal selection induced by growth factors and decrease potential treatment confounders. This resulted in an optimized version of our medium used to maintain human brain organoid cultures 12,13 . We culture tumor pieces on an orbital shaker to inc...