Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease

Abstract

AIMTo develop a human in vitro model of non-alcoholic fatty liver disease (NAFLD), utilising primary hepatocytes cultured in a three-dimensional (3D) perfused platform.METHODSFat and lean culture media were developed to directly investigate the effects of fat loading on primary hepatocytes cultured in a 3D perfused culture system. Oil Red O staining was used to measure fat loading in the hepatocytes and the consumption of free fatty acids (FFA) from culture medium was monitored. Hepatic functions, gene expression profiles and adipokine release were compared for cells cultured in fat and lean conditions. To determine if fat loading in the system could be modulated hepatocytes were treated with known anti-steatotic compounds.RESULTSHepatocytes cultured in fat medium were found to accumulate three times more fat than lean cells and fat uptake was continuous over a 14-d culture. Fat loading of hepatocytes did not cause any hepatotoxicity and significantly increased albumin production. Numerous adipokines were expressed by fatty cells and genes associated with NAFLD and liver disease were upregulated including: Insulin-like growth factor-binding protein 1, fatty acid-binding protein 3 and CYP7A1. The metabolic activity of hepatocytes cultured in fatty conditions was found to be impaired and the activities of CYP3A4 and CYP2C9 were significantly reduced, similar to observations made in NAFLD patients. The utility of the model for drug screening was demonstrated by measuring the effects of known anti-steatotic compounds. Hepatocytes, cultured under fatty conditions and treated with metformin, had a reduced cellular fat content compared to untreated controls and consumed less FFA from cell culture medium.CONCLUSIONThe 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications

“…Cells in this model remain viable and functional for at least seven days, as judged by calcein acetoxymethyl (AM) and albumin staining, respectively. The model has been used for a variety of applications, including pharmacokinetic studies, evaluation of hepatotoxicity, modeling of nonalcoholic fatty liver disease (NAFLD), and hepatitis B viral infections . In addition, a multitude of small companies and start‐ups provide liver‐on‐a‐chip platforms for which at present very limited peer‐reviewed benchmarking data are available.…”

Translating scientific discovery: the need for preclinical models of nonalcoholic steatohepatitis

“…For example, Organovo’s bioprinting platform showed evidence of steatosis (drug-induced) and fibrosis, and is being developed as a NASH model. Kostrzewski et al improved upon a previous microfluidics-based (designed by CellASIC) NAFLD model seeded with HepG2 cells, by incorporating primary human hepatocytes (no NPCs) into CN Bio’s microfluidic liver platform [28, 29]. To generate a NAFLD-relevant phenotype, both studies exposed the hepatocytes to high concentrations of oleic acid (400–660 µM) and palmitic acid (200–330 µM) combinations to create a “fat media”.…”

“…In keeping with histological observations in NAFLD patients, Kostrzewski et al reported that the fat media increased lipid accumulation in the cells and altered gene and activity levels of many CYP P450 enzymes. Cytokine levels, such as IL8, were increased in fat media conditions, but other cytokines, such as IL6 were not detected, likely due to the absence of additional cell types, such as Kupffer cells or HSCs [28]. Pioglitazone, which was found in the PIVENS trial to ameliorate fat accumulation and other histological features of NASH in the clinical setting, reduced lipid accumulation in the lipid-loaded hepatocytes, but no additional effects were reported [30].…”

Liver‐on‐a‐Chip Models of Fatty Liver Disease

“…In another FFA‐based NAFLD model, 12 individual bioreactors with a built‐in pump to control the flow of media creating an oxygen gradient were used to simulate in vivo conditions (Fig. A,B) . Devices were seeded with primary human hepatocytes and supplemented with OA and PA for 14 days.…”

“…(D) A reversibly and irreversibly injured ALD model where the development of fibrosis can be seen. Reprinted (A) with permission from Public Library of Science, (B) from Baishideng Publishing Group, (C) from John Wiley & Sons, and (D) from Royal Society of Chemistry …”