A 3D human liver tissue model of the hepatobiliary junction

Abstract

Cholestasis, or disruption in bile flow, is a poorly-understood feature of many liver diseases and is a well-established indication for liver transplant. Despite this clinical significance, many tissue engineering strategies for modeling or treating liver disease fail to recapitulate physiological bile flow. Recent advances in the field of tissue engineering and organoid technology have enabled the culture of human hepatocytes and bile duct cells in vitro, these models lack a key function of the liver which is bile transport. In this thesis, I first describe developments in bioengineering technology that have allowed for the culture and manipulation of bile duct cell organoids. I then present a 3D multicellular spheroid model that captures the structure and function of the human hepatobiliary junction—the interface between liver and bile duct cells that is often disrupted in liver disease. By co-aggregating primary human hepatocytes and bile duct cells, I engineer a liver spheroid model that recapitulates physiological bile flow through a functional connection between the two cell types. These spheroids maintain cell polarity and transport bile from hepatocyte canaliculi to bile duct structures. This function is quantified by leveraging a high-throughput imaging assay with AI-assisted analysis to track junction formation and bile flow over time. I also use this system to model ischemic injury of the bile duct, a common complication of liver transplant, by tuning the oxygen parameters of the spheroid culture. In this injury model, I observe and describe two processes that potentially contribute to injury: a reversible loss of canalicular function during hypoxia, followed by selective bile duct cell death after reoxygenation. This human-based, scalable platform provides a new tool to study bile duct biology, understand mechanisms of biliary injury after liver transplant, and support drug discovery efforts for cholestatic liver diseases.