Microfabricated Liver-on-a-Chip with Membrane-Free Endothelial-Hepatic Interface for Long-Term Culture and Drug-Induced Liver Injury Modeling.

Liver tissue function relies on cells' spatial organization and interactions within a 3D microenvironment. While previously reported liver-on-a-chip models effectively provide basic structural organization and multilayered arrangements using artificial barriers, they fall short in replicating the higher-order organization of diverse cell types. Herein, we fabricated a membrane-free liver-on-a-chip (MF-LOC) using CGRGDS peptide-modified PEGDA hydrogel that not only provides a native-like 3D microenvironment for encapsulated HepG2 (Hepatocytes) cells and NIH-3T3 (fibroblasts) but also offers a platform for HUVEC monolayer formation. The immobilization of the CGRGDS peptide on the hydrogel surface, which operates at a nanoscale level to enhance cell adhesion and signaling via integrin binding, strengthens HUVEC adhesion and prevents cell detachment caused by the shear stress of direct tangential flow. In MF-LOC, nutrients easily diffuse through the HUVEC monolayer and hydrogel pores to sustain cell functions. Live/Dead imaging and cell tracking showed HepG2 clusters associated with neighboring NIH-3T3 fibroblasts, with HUVECs forming a surface monolayer which replicate native liver structure. Functional validation confirmed prolonged albumin and urea secretion, with MF-LOC exhibiting sustained CYP1A1 enzyme activity compared to gold standard microsomes, highlighting its ability to replicate liver-like metabolism. MF-LOC has the potential for predicting drug-induced liver injury (DILI) and may provide a powerful platform for disease modeling.