Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 14, 2017; 23(2): 204-215
Published online Jan 14, 2017. doi: 10.3748/wjg.v23.i2.204
Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease
Tomasz Kostrzewski, Terri Cornforth, Sophie A Snow, Larissa Ouro-Gnao, Cliff Rowe, Emma M Large, David J Hughes
Tomasz Kostrzewski, Terri Cornforth, Sophie A Snow, Larissa Ouro-Gnao, Cliff Rowe, Emma M Large, David J Hughes, CN Bio Innovations Limited, Welwyn Garden City AL7 3AX, United Kingdom
Author contributions: Kostrzewski T performed the majority of experiments and analysed the data; Cornforth T, Snow SA, Ouro-Gnao L performed experimental analysis; Kostrzewski T, Cornforth T, Rowe C, Large EM and Hughes DJ designed and coordinated the research; Kostrzewski T and Hughes DJ wrote the paper; all authors read and reviewed the paper.
Supported by Innovate UK (Technology Strategy Board) Advancing the Development and Application of Non-Animal Technologies Project: 3D cell culture model for studying Non-Alcoholic Fatty Liver Disease (NAFLD) - Ref: 131720.
Conflict-of-interest statement: At the time of this study all authors were employees of CN Bio Innovations Ltd.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: David Hughes, DPhil, CN Bio Innovations Limited, BioPark, Broadwater Rd, Welwyn Garden City AL7 3AX, United Kingdom.
Telephone: +44-1707-358739
Received: August 16, 2016
Peer-review started: August 17, 2016
First decision: October 11, 2016
Revised: October 25, 2016
Accepted: November 16, 2016
Article in press: November 16, 2016
Published online: January 14, 2017

To develop a human in vitro model of non-alcoholic fatty liver disease (NAFLD), utilising primary hepatocytes cultured in a three-dimensional (3D) perfused platform.


Fat 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.


Hepatocytes 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.


The 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.

Keywords: Non-alcoholic fatty liver disease, Liver disease, Three-dimensional cell culture, Organ-on-chip, Primary cell culture, Fatty liver, Hepatocytes

Core tip: We report the development of an in vitro, fully human, three-dimensional cell culture model of non-alcoholic fatty liver disease (NAFLD). The model recapitulates key features of clinical NAFLD, with primary human hepatocytes continuously loaded with fat, for up to 14 d of culture, without causing any hepatotoxicity. Fat loading caused transcriptomic, proteomic and metabolic changes to the hepatocytes, including reduced activity of CYP3A4 and CYP2C9 enzymes. Fat loading in the model could be modulated using known anti-steatotic drugs (e.g., metformin), demonstrating the utility of the model for screening the efficacy of novel anti-steatotic compounds.