To optimize cell culture conditions, FLC-4 cells were cultured on two micro-patterns (200μm width X 100μm depth and 200μm width X 50μm depth). Also, surface coating and seeding density were optimized. As a result, FLC-4 formed spheroid at 7-day culture on the 200μm X 100μm pattern and the 3D structure was maintained for a week. mRNA expression of CYP1A1, 1A2, 2C9, 3A4 and UGT1A1 on 200μm X 100μm micro-space plate were higher than those in monolayer culture. mRNA expression of nuclear receptors, CAR and PXR, were also higher on micro-space culture plate than those in monolayer culture. On the other hand, spheroid formation was not observed on 200μm X 50μm micro-space plate and mRNA expression of the enzymes were much lower than those on 200μm X 100μm plate.
Enzyme induction studies showed that phenobarbital induced mRNA expression of CYP3A4 and UGT1A1 significantly on micro-space culture plate. The metabolites of triazolam and SN-38, substrates of CYP3A4 and UGT1A1, were significantly formed compared to those in monolayer culture.
These results suggest that the micro-space cell culture plates are useful tools for drug metabolism study using the human hepatocellular carcinoma cell line.