Effects of Oxygen-Permeable Plates on Cytochrome P450 and Flavin-Containing Monooxygenase in HepaRG Spheroid Cultures

Three-dimensional (3D) hepatocyte spheroid cultures offer an advanced in vitro model for investigating liver function and drug metabolism; however, oxygen diffusion limitations restrict spheroid size and functionality. In the present study, we developed a 3D spheroid culture system using HepaRG, a human hepatoma-derived cell line that retains drug-metabolizing enzymes, and oxygen-permeable plates composed of 4-polymethyl-1-pentene (PMP) to improve oxygen availability and metabolic performance. Morphological analysis showed enhanced spheroid circularity and compactness in the PMP culture, implying enhanced cell-cell adhesion. Hypoxic imaging revealed that PMP plate-based cultures markedly reduced central hypoxia, enabling the formation of spheroids with up to 20000 cells. Using clozapine, a low-clearance antipsychotic drug with limited metabolic elimination that is metabolized by monooxygenases, including CYP3A4 and flavin-containing monooxygenase (FMO), we observed an enhanced decline in clozapine levels in PMP plates compared with the clozapine levels seen in the conventional polystyrene plates. Furthermore, the mRNA and protein-expression levels of CYP3A4 and FMO3 were upregulated in the spheroids cultured on PMP plates. These results suggest that improved oxygenation enhances hepatic functionality in 3D cultures by maintaining cell viability and promoting the expression of drug-metabolizing enzymes. Therefore, the PMP plate-based spheroid model offers a practical and physiologically relevant platform for investigating drug metabolism and hepatotoxicity.