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.