A Microphysiological System for Studying Nonalcoholic Steatohepatitis

Abstract

Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments. There is an urgent need for better understanding of the genetic and molecular pathways that underlie NAFLD/NASH, and currently available preclinical models, be they in vivo or in vitro, do not fully represent key aspects of the human disease state. We have developed a human in vitro co‐culture NASH model using primary human hepatocytes, Kupffer cells and hepatic stellate cells, which are cultured together as microtissues in a perfused three‐dimensional microphysiological system (MPS). The microtissues were cultured in medium containing free fatty acids for at least 2 weeks, to induce a NASH‐like phenotype. The co‐culture microtissues within the MPS display a NASH‐like phenotype, showing key features of the disease including hepatic fat accumulation, the production of an inflammatory milieu, and the expression of profibrotic markers. Addition of lipopolysaccharide resulted in a more pro‐inflammatory milieu. In the model, obeticholic acid ameliorated the NASH phenotype. Microtissues were formed from both wild‐type and patatin‐like phospholipase domain containing 3 (PNPLA3) I148M mutant hepatic stellate cells. Stellate cells carrying the mutation enhanced the overall disease state of the model and in particular produced a more pro‐inflammatory milieu. Conclusion: The MPS model displays a phenotype akin to advanced NAFLD or NASH and has utility as a tool for exploring mechanisms underlying the disease. Furthermore, we demonstrate that in co‐culture the PNPLA3 I148M mutation alone can cause hepatic stellate cells to enhance the overall NASH disease phenotype.

Human Organs-on-Chips for Virology

“…A reliable in vitro liver model relies on reproducing the key elements of the biological processes and the physiological environment of the liver [ 29 ]. Recently, primary human liver cells, which rapidly lose function in standard culture, have been incorporated into liver chip models to study liver diseases, such as fatty liver [ 30 ], metabolic dysfunction [ 31 ], and viral hepatitis [ 32 ]. In contrast to conventional in vitro liver models, typical liver chips can include controlled ratios of various liver cell types in the correct anatomical orientation and they show improved liver-specific functionality when compared to other in vitro models.…”

Bioengineered Liver Models for Investigating Disease Pathogenesis and Regenerative Medicine

“…23 (b) Representative confocal images of PHH spheroids after induction of hepatic steatosis with free fatty acids and insulin after 14 days. 133 146 (f) Schematic approach for modeling human ALD by exposing the Liver-Chip (Emulate Inc.) to human-relevant blood alcohol concentrations (top left) and representative phase-contrast image of PHHs (top right). Fluorescent images showing lipid accumulation in PHHs visualized using AdipoRed staining after administration of fat (oleic acid 1 µg/mL; positive control) or ethanol (0.08% and 0.16%) for 48 hours (bottom).…”

“…3e). 146 The system demonstrated characteristics of a NASH-like phenotype with fat accumulation, production of inflammatory cytokines, and expression of fibrotic markers, which were elevated with the addition of lipopolysaccharide (LPS). Similar to previous models' findings, OCA alleviated the NASH-like phenotype in these cultures.…”

“…Representative images of cultures showing fat loading (Oil Red O staining, top) and quantified content (bottom left). IL-6 production in cultures with different media treatment (bottom right) 146. (f) Schematic approach for modeling human ALD by exposing the Liver-Chip (Emulate Inc.) to human-relevant blood alcohol concentrations (top left) and representative phase-contrast image of PHHs (top right).…”

Recent advances in phenotypic drug discovery

“…Unfortunately, the complexities of biology preclude a one-size-fits-all solution; each approach with their strengths and weaknesses must be individually considered (reviewed by 39 , 40 ) with the goal of optimizing issues related to assay enablement, testing capacity, operational costs, and clinical relevance of the assay. In principle, the chain of translatability of a cellular system should be benchmarked against multiple aspects of the human disease state 43 .…”