Establishment of a cell culture platform for human liver organoids and its application for lipid metabolism research

Human liver organoids (HLOs) are reliable tools to represent physiological human liver biology. However, their use is limited especially in basic sciences. One of the reasons for this would be the insufficient systematic methodology to handle HLOs, including culture system, functional assessment, and gene transduction. Here, we generated and characterized mouse L cells stably and simultaneously overexpressing R-spondin1, hepatocyte growth factor, fibroblast growth factor (FGF) 7, and FGF10 via lentiviral transduction. The conditioned medium of the cells contributed to HLO growth as a replacement of commercially available recombinant proteins, which leads to a significant reduction of their culture cost. Proliferative and maturation phases of the cells were controlled by switching the medium to facilitate the evaluation of hepatocyte function, including insulin responsiveness and intracellular lipid accumulation. Gene expression analysis revealed that HLOs highly expressed genes involved in lipid metabolism. Importantly, HLOs secreted physiologically matured very low-density lipoprotein, which is rarely observed in mice and in established cell lines. Efficient gene transduction into HLOs was achieved via a transient 2-dimensional culture during viral infection. This study provides an invaluable platform for utilizing HLOs in various research fields, such as molecular biology, pharmacology, toxicology, and regenerative medicine. Human liver organoids (HLOs) can reflect human liver physiology; however, methods to use HLOs are insufficient. Here, the authors generated L cells overexpressing essential factors in a balanced manner to prepare an expansion medium for HLOs, confirmed the physiological function of HLOs, and achieved efficient gene transduction into HLOs. This work provides a fundamental platform for handling HLOs in basic and applied sciences.image