Development of In Vitro NASH Model with Mechanically Compliant Substrate

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Although a sedentary lifestyle is known to play a role in nonalcoholic steatohepatitis (NASH), understanding of the molecular mechanism of NASH development is incomplete and effective treatment is still greatly sought. Part of this challenging situation in tackling NASH is a lack of an appropriate in vitro hepatocyte culture that reproduces in vivo cellular functions ex vivo. Emerging evidence suggests that matching the stiffness of substrates with that of tissues helps reproducing in vivo functions of cells derived from them. Thus, we hypothesized that primary hepatocytes on 500 Pa polyacrylamide (PAA) gels, whose stiffness matches that of the liver, may exhibit steatosis and a pro-inflammatory response when treated with either fructose or palmitate to model over-nutrition. To this end, rat primary hepatocytes were seeded on either 500 Pa PAA gels or glass coverslips, which represent a conventional but unphysiological stiff culture device. Cells were cultured in low glucose Dulbecco’s Modified Eagle’s medium, whose glucose concentration matches the normal blood glucose level, with or without addition of 5.5 mM fructose or 0.5 mM palmitate. Addition of fructose or palmitate induced lipid accumulation in cells on 500 Pa gels and increased ROS (reactive oxygen species) accumulation in hepatocytes on 500 Pa PAA gels, but not in hepatocytes on glass. Fructose or palmitate treatment also caused HMGB1 (High Mobility Group Box 1) secretion by hepatocytes, implicating a proinflammatory response by these cells. These results suggest that exposure of hepatocytes on a mechanically compliant substrate to either fructose or palmitate may serve as an excellent ex vivo model of the initial phase of NASH development by showing ability to initiate an inflammatory process. Disclosure M. Morishima: None. K. Horikawa: None. M. Funaki: Research Support; Self; Otsuka Holdings Co., Ltd., The Knowledge Cluster from the Ministry of Education, Science, and Culture of Japan. Board Member; Self; Mechanogenic C.C..