In Vitro and In Vivo Models of Non-Alcoholic Fatty Liver Disease: A Critical Appraisal

Abstract

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), represents the hepatic manifestation of obesity and metabolic syndrome. Due to the spread of the obesity epidemic, NAFLD is becoming the most common chronic liver disease and one of the principal indications for liver transplantation. However, no pharmacological treatment is currently approved to prevent the outbreak of NASH, which leads to fibrosis and cirrhosis. Preclinical research is required to improve our knowledge of NAFLD physiopathology and to identify new therapeutic targets. In the present review, we summarize advances in NAFLD preclinical models from cellular models, including new bioengineered platforms, to in vivo models, with a particular focus on genetic and dietary mouse models. We aim to discuss the advantages and limits of these different models.

Keywords: NAFLD; NASH; cell culture; liver-on-a-chip; mouse model; organoids; spheroids.

Figure 1

Hepatic cell sources and in vitro models of non-alcoholic fatty liver disease (NAFLD). Different cell sources are now available to build in vitro models of NAFLD. Primary human hepatocytes (PHH) display the closest phenotype to the human liver. A short culture time and low availability of PHHs are alleviated by using immortalized cell lines (HepG2, HuH7 or HepaRG) and hepatocyte-like cells (HLC) derived from stem cells (as human-induced pluripotent cell—hIPSC, embryonic stem cells—ESC or hepatic stem cells—HSC, or mesenchymal stem cells—MSC). However, altered metabolic functions and incomplete differentiation of HLC are limitations of the model. Several models have been developed to mimic NALFD in vitro. NAFLD can be studied in 2- or 3-dimensional cell cultures by adding free fat acid (FFA), Bisphenol A or Valproate in the culture medium. New 3D models have been recently developed: spheroids are derived from different hepatic cell sources; hepatic organoids are produced by stem cell differentiation in parenchymal and non-parenchymal liver cells. Livers-on-a-chip are devices designed to mimic the physiological environment of the liver lobule: hepatocytes are placed inside a micro-scaffold, and a fluidic flow passes through the chip to reproduce blood circulation.

Figure 2

Mouse models of NAFLD. Several mouse models have been described to mimic NAFLD in vivo. Both genetic and diet models are widely used to create experimental conditions of NAFLD and for preclinical drug testing. However, each model is specific as mice do no present all the typical features of NAFLD, from non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). High-fat diet (HFD) and Western diet-fed mice, as well as genetic ob/ob (leptin deficiency) and db/db (leptin receptor mutation) mouse models, display metabolic syndrome and severe steatosis but no liver inflammation or fibrosis. However, the Methionine- and Choline-deficient diet (MCD) and its derivate diets such as Choline Deficient Ethionine-supplemented (CDE) and Choline-deficient L-amino acid-defined (CDAA) diets promote liver inflammation and fibrosis. The stelic animal model (STAM) associates a toxic injection of streptozotocin with an HFD, resulting in HCC development. Other genetic models, such as Melanocortin-4 receptor (MC4R) deficiency or Sterol regulatory element-binding proteins (SREBP1c) overexpression, predispose to metabolic syndrome and obesity but need a second hit (such as HFD) to trigger NASH.