2.7. Spheroid formation assay

T98G and A172 cells were cultured in spheroid culture medium containing 1.6% (w/v) methylcellulose (Nacalai Tesque). Fresh aliquots of MACS NeuroBrew‐21, EGF and bFGF were added at day 3. After 6 days in culture, the living cells were stained with Calcein‐AM (Dojindo) and counted using hybrid cell count module (Keyence). In some experiments, cells were treated with Strictinin (Nagara Science Co., Ltd.), a Ror1 inhibitor. The numbers and sizes of spheroids formed were measured, and spheroids were classified into small (<1000 μm²), medium (1001–2000 μm²), and large (>2001 μm²) according to their sizes.