Penicillin streptomycin amphotericin b mixture

Tumor tissues were surgically obtained from the site of the tumor periphery of four GBM patients and were mechanically minced, digested with 0.1% trypsin, and then triturated with a Pasteur pipette. Cells were passed through a 70 μm strainer (Falcon; Becton Dickinson Biosciences) and resuspended in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin/amphotericin B mixture (Wako). Ability of the tumor cells in the primary cell culture to generate a sphere formation was examined by placing the cells in serum-free DMEM/Ham's F-12 (Wako) containing 10 μg/ml insulin (Wako), 10 nmol/l recombinant human basic fibroblast growth factor, 10 nmol/l recombinant human epidermal growth factor, 5 μmol/l heparin, N2 supplement (Wako), GlutaMAX supplement (GIBCO), and the penicillin/streptomycin/amphotericin B mixture (neural stem cell medium). Growth factors were purchased from PeproTech, London, UK. Primary tumor cells that were cultured in DMEM with 10% FBS, from which the sphere-forming cells (SFCs) were obtained by replacing the medium, were named as parent cells (PCs) for each SFCs.