Protocol to establish glioma stem cell spheroids and an enriched cancer-associated fibroblast population from a single patient tumor specimen

Glioblastoma is an aggressive, therapy-resistant brain tumor in which the role of cancer-associated fibroblasts (CAFs) remains poorly defined. We present a protocol to simultaneously establish glioblastoma stem cell (GSCs) spheroids and CAFs from a single patient specimen. We describe steps to dissociate tumor tissue, generate a single-cell suspension, and establish GSCs spheroids and CAFs cultures. This protocol details CAFs enrichment, passaging, phenotypic characterization, and cryopreservation. For complete details on the use and execution of this protocol, please refer to Delmas et al. 1 • Standardized protocol to isolate GSC spheroids and enriched CAFs from single GBM sample • Steps for tumor dissociation and generation of GSC spheroids and CAF cultures • Procedure for CAF enrichment, passaging, characterization, and cryopreservation • Procedure for GSC culture, passaging, characterization, and cryopreservation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Glioblastoma is an aggressive, therapy-resistant brain tumor in which the role of cancer-associated fibroblasts (CAFs) remains poorly defined. We present a protocol to simultaneously establish glioblastoma stem cell (GSCs) spheroids and CAFs from a single patient specimen. We describe steps to dissociate tumor tissue, generate a single-cell suspension, and establish GSC spheroids and CAF cultures. This protocol details CAF enrichment, passaging, phenotypic characterization, and cryopreservation.