Establishment of an ex Vivo Model of Nonalcoholic Fatty Liver Disease Using a Tissue-Engineered Liver

The prevalence of nonalcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease, continues to increase in parallel with that of obesity. Currently, there are no preclinical models to study its complex pathogenesis nor to assess candidate therapies. We have established a tissue-engineered (TE) liver by seeding cells into liver-derived matrix scaffolds and then perfusing the scaffolds with a medium that dynamically provides requisite nutrients, vitamins, minerals, and hormones. Liver-specific biomatrix scaffolds, comprised of almost all of the liver's known extracellular matrix (ECM) components and matrix-bound soluble signals (e.g., growth factors/cytokines), were recellularized with human hepatic cell line HepG2 and perfused with a complete medium enabling the cells to form functioning liver tissue. By perfusing the system with medium with a high fat content, the cells established a TE fatty (TEF) liver model paralleling that of livers in NAFLD patients. The high fat medium containing 500 μM of free fatty acids (FFAs) (oleic acid:palmitic acid = 2:1) caused the TEF livers to accumulate 2-times more fat than those in the control medium over an 8 day culture period and significantly influenced the capacity of fatty acid synthesis and metabolism. PDK4, CYP2E1, and CYP7A1 genes associated with NAFLD and other liver diseases were all up-regulated, and the metabolic activity of CYP3A4 was significantly impaired. Excess FFAs also induced alterations in transporters and key enzymes in the lipid biosynthesis pathway. The TEF liver was used to test if an antisteatotic drug, Metformin, used in patients with NAFLD, would be able to provide effects paralleling those observed in some patients. Metformin treatment of the TEF liver model caused reduced cellular triglycerides, activated AMPK molecule, inhibited mTORC1 signaling pathway, which thus affected the synthesis and metabolism of FFAs. Overall, the TEF liver offers a stable and reproducible model to study the NAFLD development process and antisteatotic drug effects.