A 3D In Vitro Model of the Human Hepatobiliary Junction

ABSTRACT Bile flow is an essential feature of the liver, and disruption of this process contributes to a range of liver pathologies. Efficient bile transport requires coordinated organization between hepatocytes and cholangiocytes at the hepatobiliary junction, a structure that remains poorly captured in existing in vitro models of liver disease. Here, we present a 3D multicellular spheroid‐based model of the human hepatobiliary junction. Building on advances in organoid and spheroid engineering, we co‐aggregate human hepatocytes and intrahepatic cholangiocytes, supported by murine fibroblasts, into adult hepatobiliary organoids (aHBOs). aHBOs directionally transport bile from hepatocyte canaliculi to cholangiocyte‐lined ductule‐like structures, visualized through a high‐throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using a fluorescent bile acid analog and AI‐assisted image analysis. When subjected to hypoxia‐reoxygenation, aHBOs exhibit disrupted bile transport and distinct cell‐type‐specific responses, enabling interrogation of hepatocyte and cholangiocyte vulnerability to transplant‐associated biliary hypoxia. Our findings suggest a reversible reduction in hepatocyte canalicular function under hypoxia, followed by selective cholangiocyte death upon reoxygenation, potentially contributing to biliary dysfunction after ischemic injury. This human‐derived, scalable platform provides a phenotypically relevant model for dissecting mechanisms of biliary dysfunction and discovering therapeutics for hypoxic liver injury and cholestatic diseases.