Establishment of Glioma Cell Cultures and Transduction

RCAS/tv-a system has been described in previous studies25 (link)26 (link). PDGF-induced gliomas were dissected and enzymatically digested for 10 minutes in EBSS solution containing 12% papain (Worthington) and 10 μg/ml DNase at 37 °C. The digestion was stopped using 1 mg/ml ovomucoid (Worthington) dissolved in basal neural medium containing 10 μg/ml DNase. Cells were washed using basal neural medium three times and plated in DMEM containing 10% FBS. Only the cultures between passage numbers p0 and p2 were used in the study. U87-MG cells were obtained from ATCC. U3020-MG is a primary human GBM line from HGCC (www.hgcc.se), as previously described11 (link). To generate ABCG2-expressing cultures, Platinum E packaging cells were transfected with retroviral constructs, pbabe-EMPTY-puro and pbabe-ABCG2-puro, that were previously described7 (link) using Fugene (Roche). Viral supernatants were then harvested 24 hrs after transfection and filtered using 0.45 μm filter, and cells were infected for two cycles for two days in the presence of 8 μg/ml of polybrene. Cells were recovered for 24 hrs or until they reached 70–90% confluency with fresh medium, and then selected with 1 or 2 μg/ml puromycin depending on cell lines. Cell viability was measured by the MTT assay (Roche) as per the manufacturer’s recommendations.