Engineering physiologically relevant In vitro liver models for inflammation response and vascularized co-culture

Abstract

In vitro human liver models are becoming increasingly crucial for disease modeling and drug development. Here we take a design principle-driven approach to engineering more physiologically relevant 3D liver models by focusing on microenvironmental parameters influencing physiological functions. Specifically, we address the biophysical properties of the scaffold and its interplay with culture medium components and examine the effects on phenotypes, including inflammation response. First, we engineered a poly(ethylene glycol) (PEG)-based hydrogel scaffold to support 3D tissue formation in a flow-driven bioreactor. This geometrically controllable, low swelling, and physiologically stiff gel sustained a healthier hepatic morphology and better functional stability compared to traditional polystyrene cultures. Second, we improve upon existing hepatic 3D liver spheroid aggregation platforms by engineering a 3D-printed alginate microwell system.

Spheroids produced with this microwell system are functionally stable in long-term culture and can be efficiently harvested for downstream applications. We investigated liver models generated from these PEG and alginate scaffolds for their response to inflammatory signals in culture media. We initially observed a lower basal inflammatory state when primary human hepatocytes were cultured in both systems compared to polystyrene. We then developed a defined, serum-free growth factor supplemented medium, which enhanced hepatocyte function and strongly induced an inflammatory and regenerative microenvironment. Gene expression profiling, multiplexed cytokine analysis, and imaging indicated that differential responses in each scaffold were due to the attenuation of YAP/TAZ signaling. Notably, PEG scaffolds gave the highest response range, indicating that this model can respond dynamically to stimuli.

Additionally, we saw a more controlled dose-dependent response in PEG to short-term TGF[beta] stimulation. Using the culture technologies and the developed media, we engineered a vascularized tri-culture model using hepatocytes, endothelial cells, and mesenchymal stem cells. This model forms a robust vascular network that interacts with hepatic spheroids. Seeding tri-culture spheroids into a flow-driven bioreactor resulted in the formation of lumen-like features indicative of physiological polarization and morphology. This thesis illuminates design principles for multifaceted liver tissue engineering and introduces generalizable and translatable technologies. We further enable the design of sophisticated liver disease models that can enable more predictable drug development and insights into liver biology.