Westerfield Ashley D, Grzelak Katarzyna A, Katsuyama Katie, Kumar Vardhman, Miller Bess M, Yun Joa, Kirkpatrick Jesse, Mankus David, Bisher Margaret E, Lytton-Jean Abigail K R, Jiang Z Gordon, Lee David D, Chen Christopher S, Bhatia Sangeeta N
bioRxiv. 2025 Jul 14:2025.07.11.664464. doi: 10.1101/2025.07.11.664464.
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 model for dissecting biliary pathophysiology and lays the groundwork for a therapeutic discovery platform for post-transplant ischemic cholangiopathy and other cholestatic liver diseases.
胆汁淤积，即胆汁流动中断，是许多肝脏疾病和损伤中常见但了解不足的特征。尽管如此，许多工程化的人类肝脏疾病组织模型未能重现生理性胆汁流动。在此，我们展示了一种基于三维多细胞球体的人肝胆交界处模型，该交界处是肝细胞和胆管细胞之间的界面，在肝脏疾病中常被破坏，而它对于引导肝细胞分泌的胆汁进入胆管系统是必需的。基于类器官和球体工程的进展，我们将人肝细胞和肝内胆管细胞共聚集形成成人肝胆类器官（aHBOs），其在结构上相互连接并在功能上运输胆汁。aHBOs将胆汁从肝细胞胆小管定向运输到内衬胆管细胞的小导管，我们通过高通量成像分析对其进行可视化。使用荧光胆汁酸类似物和人工智能辅助图像分析对肝胆交界处的形成和胆汁流动动力学随时间进行量化。当受到缺氧 - 复氧处理时，aHBOs重现了胆汁功能障碍的特征，模拟了肝移植中并发的胆汁淤积和缺血 - 再灌注损伤。我们的数据表明：1）缺氧时肝细胞胆小管功能的可逆性降低，随后2）复氧时胆管细胞的选择性死亡，是缺血性损伤后可能导致胆汁功能障碍的过程。这个源自人类的、可扩展的平台为剖析胆汁病理生理学提供了一个表型相关的模型，并为移植后缺血性胆管病和其他胆汁淤积性肝病的治疗发现平台奠定了基础。
