Serum-free medium (no FBS) FGF-2 and EGF are required (stem cell medium) (Lee et al., 2006; Claes et al., 2008) Grown in low-attachment conditions (i.e., no laminin-coated plates in case of glioma CSCs) Low seeding density to avoid aggregation and to foster clonal expansion. Here, FGF2 is linked to glioma.