摘要：SummarySpheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. The cultures are maintained under different conditions, with possible confounding results. We performed an in-depth analysis of the influence of various culture conditions to find the optimal conditions for the maintenance of anin vivolike phenotype. The formation, protein expression, and function of PHH spheroids were followed for three weeks in a high-throughput 384-well format. Medium composition affected spheroid histology, global proteome profile, drug metabolism and drug-induced toxicity. No epithelial-mesenchymal transition was observed. Media with fasting glucose and insulin levels gave spheroids with phenotypes closest to normal PHH. The most expensive medium resulted in PHH features most divergent from that of native PHH. Our results provide a protocol for culture of healthy PHH with maintained function - a prerequisite for studies of hepatocyte homeostasis and more reproducible hepatocyte research.Graphical abstractDisplay OmittedHighlights•3D spheroid cultures were established in 384-well format•Eight different media variants were used to optimize the 3D cultures•Optimized William's medium was as good as expensive commercial medium•The 3D cultures were used to study drug metabolism and toxicityBiological sciences; Proteomics; Methodology in biological sciences
