Fgf fibroblast growth factor

CT-2A cells were provided by Prof. Thomas Seyfried (Boston College, Boston, MA, USA) (Seyfried et al., 1992 (link)). Cells were incubated at 37°C in humidified air with 5% CO₂. ML/CT-2A cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific), supplemented with 10% heat-inactivated fetal calf serum (FCS; Thermo Fisher Scientific) (Seyfried et al., 1992 (link)). To generate NS/CT-2A cells, we adapted a previously published protocol (Binello et al., 2012 (link)). Briefly, confluent ML were enzymatically dissociated (Stempro Accutase, Thermo Fisher Scientific) and cells were plated at 1×10⁵ cells/ml in DMEM/Nutrient Mixture F-12 (DMEM/F-12; Thermo Fisher Scientific) supplemented with 20 ng/ml epidermal growth factor (EGF; Thermo Fisher Scientific), 20 ng/ml fibroblast growth factor (FGF; Thermo Fisher Scientific) and 2% B27 supplement (Thermo Fisher Scientific). Six days after plating, NS were collected, enzymatically dissociated and re-plated at the same initial concentration. Two and 8 days after the start of the NS culture, the medium was supplemented with 20 ng/ml EGF and FGF. Eleven days after the start of the culture, NS were collected, enzymatically dissociated and prepared for further applications.

Human glioblastoma cell lines U87MG and A172 and the human fetal glial cells SVG-P12 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and Pt#3 was derived from a GBM patient [24 (link)]. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and penicillin/streptomycin (Thermo Fisher Scientific) in a humidified atmosphere containing 5% CO₂. For spheroid cell culture, cells were cultured in serum-free DMEM/F12 (Thermo Fisher Scientific) supplemented with 100 μM sodium pyruvate, 10 ng/mL epidermal growth factor (EGF), 10 ng/mL fibroblast growth factor (FGF) (Thermo Fisher Scientific), and N-2 supplement (Thermo Fisher Scientific) in the culture dishes pre-coated with poly-2-hydroxyethyl methacrylate (poly-HEMA).