T flasks

ReNcell-VM (Millipore®) is a human neural progenitor cell line derived from the ventral mesencephalon region of the fetal brain and immortalized by retroviral transduction with the v-myc oncogene. ReNcell-VM were first expanded in T-flasks (Falcon®, Corning) previously coated with poly-L-ornithine (PO) (20 μg mL⁻¹, Sigma-Aldrich) for 30 min and laminin (LN) (10 μg mL⁻¹ in PBS, Sigma-Aldrich) for 4 h at 37°C and 5% CO₂. After seeding, cells were expanded in N2 medium, consisting of DMEM/F12 with glucose (1.6 g L⁻¹, Sigma-Aldrich), N2-supplement (1%, Thermo Fisher), penicillin/streptomycin (1%, Thermo Fisher) and insulin (20 μg mL⁻¹, Sigma-Aldrich), and supplemented with EGF (20 ng mL⁻¹, Peprotech), FGF-2 (20 ng mL⁻¹, Peprotech), and B27 supplement (20 μl mL⁻¹, Thermo Fisher).