Abstract
Cholestasis, or disruption in bile flow, is a common yet poorly understood feature of many liver diseases and injuries. Despite this, many engineered human tissue models of liver disease fail to recapitulate physiological bile flow. Here, we present a 3D multicellular spheroid-based model of the human hepatobiliary junction, the interface between hepatocytes and cholangiocytes often disrupted in liver disease that is required for directing bile excreted by hepatocytes into the biliary ductal system. Building on advances in organoid and spheroid engineering, we co-aggregate human hepatocytes and intrahepatic cholangiocytes into adult hepatobiliary organoids (aHBOs) that structurally connect and functionally transport bile. aHBOs directionally transport bile from hepatocyte bile canaliculi to cholangiocyte-lined ductules, which we visualize through a high-throughput imaging assay. Hepatobiliary junction formation and bile flow dynamics are quantified over time using fluorescent bile acid analogs and AI-assisted image analysis. When subjected to hypoxia-reoxygenation, aHBOs recapitulate features of biliary dysfunction that mimics the cholestasis and ischemia-reperfusion injury that complicates liver transplant. Our findings suggest that 1) a reversible reduction in hepatocyte canalicular function under hypoxia, followed by 2) selective cholangiocyte death upon reoxygenation, are processes that potentially contribute to biliary dysfunction upon ischemic injury. This human-derived, scalable platform provides a phenotypically-relevant in vitro model for dissecting biliary pathophysiology and lays the groundwork for a therapeutic discovery platform for post-transplant ischemic cholangiopathy and other cholestatic liver diseases.