Culturing and Expanding Patient-Derived GBM Spheres

The human U-87 MG (ATCC^® HTB-14™) (ATCC, Manassas, VA, USA) and GBM8401 GBM cell lines used in the study were purchased from (Bioresource Collection Research Center, Hsinchu, Taiwan). The cell lines were cultured in Gibco DMEM (Cat. No. 11965175, Thermo Fisher Scientific, Inc. Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen, Life Technologies, Carlsbad, CA, USA) and incubated in 5% humidified CO2 incubator at 37 °C. The cells were sub-cultured when they reached 80–90% confluency and the media changed every 48–72 h. Patient-derived CD133 + GBM spheres were kindly provided by our collaborator Dr. Alexander T.H. Wu at Taipei Medical University. In brief, the patient-derived GBM cells were first sorted using the established flow cytometric method. Once CD133+ cells were sorted, they were expanded in advanced DMEM/F12 (Gibco) mixed with Neurobasal TM-A medium (Gibco) (1:1) supplemented with B-27 (1×), FGF (20 ng/mL) and EGF (20 ng/mL); culturing under these conditions maintained CD133+ cell population and stemness (as well as TMZ-resistant), the tumor-initiating ability was demonstrated in vivo as described previously [35 (link)].