Chemical‐based primary human hepatocyte monolayer culture for the study of drug metabolism and hepatotoxicity: Comparison with the spheroid model

Abstract Traditionally cultured monolayers of primary human hepatocytes (PHHs) deteriorate within days and thereby become unsuitable for drug‐related studies. PHH spheroids (3D PHHs) maintain liver functions for weeks, but are considerably more demanding. Recently, a chemical‐based approach (5C PHHs) succeeded in long‐term culture of hepatocyte monolayers, but it remains unclear whether the drug‐related functions are preserved. To clarify this, we compared the 5C and 3D PHHs in terms of gene expression analysis, proteomic analysis, functionality (basal and induced activities of representative CYP450 enzymes and urea and albumin secretions), survival in culture, and sensitivity to representative drugs. In all comparisons, which spanned culture durations of up to 4 weeks, the 5C PHHs performed at least as well as the 3D PHHs. Hence, the novel 5C PHH monolayer format combines the convenience of the traditional monolayer format with the functionality and maintainability of the spheroid format. Our results suggest that 5C PHH monolayers can be used more conveniently and efficiently for high‐throughput drug screening, preclinical drug safety evaluations, and mechanistic studies.