Primary brainstem gliomas were digested at 37°C for 15 minutes in dissociation solution containing EBSS, 0.94 mg/mL papain (Worthington); 0.18 mg/mL EDTA, 0.18 mg/mL cysteine; and 0.06 mg/mL deoxyribonuclease I (DNase). After extensive trituration, the glioma cells were pelleted and resuspended in an ovomucoid solution, containing 0.7 mg/mL ovomucoid (Worthington) and 0.01 mg/mL DNase in NeuroCult basal medium (STEMCELL Technologies). The glioma cells were cultured in DMEM with high glucose and pyruvate (Gibco) supplemented with 10% FBS, 2 mM l-glutamine, and 1% antibiotic-antimycotic (Gibco). Cells were passaged at least 5 times to enrich for tumor cells and deplete stromal cells.

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