Isolation and expansion of regionally defined human glioblastoma cells in vitro.

This unit describes a protocol for the surgical collection, isolation, and expansion of regionally defined human glioblastoma multiforme (GBM)-derived cells. Given the important role played by microenvironmental hypoxia in defining cell phenotype and molecular signaling activation, it is important to distinguish between tumor tissues that were originally localized within the partially necrotic tumor core and those located along the peripheral and vascularized areas. The procedures for enzymatic dissociation of GBM tissues and cell culturing under hypoxia described here are optimized to obtain an efficient single-cell suspension and subsequent growth, in an effort to avoid the spontaneous induction of cell commitment normally occurring in long-term cell culture. John Wiley & Sons, Inc.