Isolation and Culture of Primary Glioblastoma Cells from Human Tumor Specimens

Cultured tumor cells are a central tool in cancer research and have provided fundamental insights in tumor biology. Recent evidence, however, indicates that classically established cell lines from different tumors, including glioblastoma, do not fully reflect the genotypes and phenotypes of the respective primary tumors. By contrast, primary cells, isolated from human tumor samples and maintained in serum-free spheroid cultures at low passage under defined growth factor conditions, reproduce key aspects of tumor cell physiology much more faithfully. Among the tumor cell characteristics that are better represented in primary glioblastoma cell cultures is the self-renewal and differentiation potential of the tumor cells. Indeed, a large body of evidence from the past decade indicates that glioblastomas and other tumors are composed of a hierarchy of heterogeneous types of cells, which are generated and maintained by cells that share characteristics of stem cells. This cancer stem cell/tumor initiating cell population is optimally preserved and maintained in primary glioblastoma cultures. Here, we describe a method for the isolation and culture of primary tumor cells from human glioblastomas in serum-free conditions, which allows the routine generation and proper maintenance of tumor cells as spheroid cultures. Such primary tumor cultures can serve as a model of choice for the study of the mechanisms behind key aspects of glioblastoma biology, including tumorigenicity, stem cell hierarchy, invasion, and therapeutic resistance.