2.8. 3D Spheroid Culture

Spheroids were established by using the liquid overlay method. First, 50 µL of 1.2% (in 95% ethanol) poly-HEMA (polyhydroxyethylmethacrylate) were added to each well of 96-well round bottom plates and left to dry at 37 °C for two days. A431 squamous cell carcinoma cells or normal human skin BJ fibroblasts were seeded at a density of 5 × 10⁴ cells/well in 200 µL medium DMEM/F-12, 1% streptomycin, and 10% FBS. After 5 days of spheroid formation, the supernatant was cautiously substituted with fresh culture medium prior to treatment [17,28].