A Dynamic 3D Human Liver Sinusoid Model for Mechanistic Interrogation of Fontan‐Associated Liver Disease

Fontan-associated liver disease (FALD) is a progressive complication of Fontan circulation, driven by chronically elevated central venous pressure (CVP) and hypoxia. Current experimental models lack the fidelity to fully recapitulate the complex hemodynamic and cellular microenvironment of the liver sinusoid under Fontan physiology, limiting mechanistic insights and therapeutic discovery. We developed a perfusable, 3D bioengineered human liver sinusoid model incorporating hepatocytes and endothelial cells within a multilayered construct. The platform was cultured under tunable flow and oxygen, simulating physiological and pathological CVP. Structural and mechanical fidelity were assessed using electron microscopy and microindentation. Hemodynamic measurements by catheter and particle image velocimetry were in agreement with those predicted by computational modeling. Sinusoid analogues maintained their architecture, endothelial coverage, and hepatic viability and function over 21 days of culture. Elevated pressure and hypoxia resulted in endothelial activation (VCAM-1, ET-1), hepatocellular stress (HIF1α), fibronectin-rich tissue remodeling, and altered hepatic function (albumin, bile acid, LDH, TGF-β), consistent with early FALD pathology. Structural and molecular responses were altered with varying pressure and oxygen, confirming the platform's sensitivity to mechanical and metabolic cues. This bioengineered 3D model successfully reproduced the key early features of FALD pathophysiology, enabling controlled interrogation of pressure- and hypoxia-induced liver injury.