Protocol for in vitro establishment of heterogeneous stem-like cultures derived from whole human glioblastoma tumors

Cultures enriched in glioblastoma stem-like cells (GSCs) are prominent in vitro models to investigate molecular determinants and therapeutic targets of glio-blastoma; however, conventional GSC derivation protocols fail to preserve GSC heterogeneity. Here, we present a protocol for the propagation of hetero-geneous GSC cultures starting from cell resuspensions containing the entire tu-mor mass. We describe steps for isolation of GSCs and their maintenance and expansion in culture. We then detail procedures for preliminary analysis to be performed on freshly isolated material. For complete details on the use and execution of this protocol, please refer to De Bacco et al.1