Figure 1.

HepG2 spheroid culture and exposure scheme. One thousand viable cells were seeded into 1.5% agarose-coated 96-flat-bottom well plates and were cultivated at a 37 °C humidified incubator with 5% CO₂ and maintained for 14 days. The culture medium was refreshed every 3–4 days. Treatments started on D7 (mean diameter of spheroids: 250–300 μm). Treatment was performed every second to third day, and the hepatospheroids were repeatedly exposed for 7 days (three exposures). The concentration of DMSO was maintained at 0.01% at all experimental conditions. Spheroid growth was monitored using a phase-contrast microscope (BioTek) during every treatment duration.