A. Cell culture

Glioma GL261 mouse cells are cultured at 37  °C under 95% air/5% CO₂ atmosphere in culture medium consisting of Dulbecco's modified eagle medium (DMEM) enriched with 10% calf serum and 5% penicillin streptomycin (Penstrep). Cell spheroids are prepared from confluent cell cultures using the suspension-spinning method. Spheroids ranging from 80 to 450 μm in diameter are obtained from 5 ml of cell suspension in CO₂-equilibrated culture medium at a concentration of 4×105 cells per ml after placement in a gyratory orbital shaker at 75 rpm at 37  °C for 30–50 h. The flasks are pretreated with 2% dimethylchlorosilane in chloroform to prevent adhesion of cells to the glass surface.