Characterization of human intestinal organoid-derived monolayers under air-liquid interface culture for...

Intestinal organoid-derived monolayers have emerged as a novel in vitro model for investigating drug absorption and metabolism. Air-liquid interface (ALI) culture method enhances physiologically relevance of epithelial cells. In this study, we applied ALI culture to tissue stem cell-derived human intestinal organoids with the aim of achieving further improvement of their pharmacokinetic function. We established human duodenal and jejunal tissue-derived organoid monolayers under ALI culture using commercially available ready-to-use media and evaluated their utility for their pharmacokinetic application in comparison with conventional submerged monolayers. Although transepithelial electrical resistance values were significantly lower and more stable in ALI monolayers, monolayers under both conditions maintained barrier integrity as indicated by low apparent permeability of fluorescein-isothiocyanate dextran 4000, a paracellular transport marker. ALI monolayers exhibited markedly higher expression of CYP3A4 along with enhanced CYP3A activity than submerged monolayers. Correspondingly, an estimated intestinal availability value of midazolam, a CYP3A substrate, in ALI monolayers closely matched a reported human in vivo value, outperforming submerged monolayers. Activities of phase II metabolic enzymes (uridine 5'-diphospho-glucuronosyltransferase and sulfotransferases) and P-glycoprotein, as well as CYP3A4 and P-gp inducibility, were comparable between the culture conditions. Our findings demonstrate that ALI-cultured human intestinal organoid-derived monolayers provide a robust in vitro platform for investigating intestinal first-pass metabolism of CYP3A substrate drugs. SIGNIFICANCE STATEMENT: This study provides a detailed comparison of tissue-derived human intestinal organoid monolayers cultured under air-liquid interface (ALI) and conventional submerged conditions, focusing on their applicability in pharmacokinetic studies. Our findings highlight the enhanced capability of ALI monolayers to recapitulate CYP3A-mediated intestinal first-pass metabolism. Leveraging commercially available ready-to-use organoid culture media, the ALI model offers a practical and efficient platform for drug metabolism studies, facilitating broader adoption depending on specific research needs.