Establishing primary glioblastoma cell cultures

The histological grade of the surgical specimen from a patient with WHO grade IV glioblastoma was established by histopathological analysis. The tissue sample was collected during surgery and immediately processed. First, the tissue was chopped with a surgical blade in a sterile Petri dish. Chemical dissociation was achieved by adding Accumax solution (Sigma-Aldrich Chemie GmbH) to the chopped tissue for 15 min at room temperature. Dissociated tissue was then centrifuged and resuspended in 5 mL of DMEM/F12 medium, supplemented with 10% FBS, 2 mM L-glutamine, 10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin B solution. DMEM/F12 medium was additionally supplemented with growth factors: 20 µL/mL B-27, 40 ng/mL epidermal growth factor and 20 ng/mL basic fibroblast growth factor, all purchased from Thermo Fisher Scientific. To confirm cell attachment, dissociated tissue was monitored for 48 h before changing the medium. Attached cells were grown until confluence prior to further studies. The glioblastoma origin of the established primary culture, named GBM-6, was confirmed as previously described [25 (link)]. The passage number of GBM-6 was kept low to prevent genetic and epigenetic changes in cells [28 (link)], to ensure the translational potential of obtained results [29 (link)].