A 3D Human Liver Model of Nonalcoholic Steatohepatitis

Marion Duriez , Agnes Jacquet , Lucile Hoet , Sandrine Roche , Marie-Dominique Bock , Corinne Rocher , Gilles Haussy , Xavier Vigé , Zsolt Bocskei , Tamara Slavnic , Valérie Martin , Jean-Claude Guillemot , Michel Didier , Aimo Kannt , Cécile Orsini , Vincent Mikol , Anne-Céline Le Fèvre 
Source: J Clin Transl Hepatol
Publication Date: (2020)
Issue: 8(4): 359-370
Research Area:
Drug Discovery
Cells used in publication:
Hepatocyte, human
Species: human
Tissue Origin: liver
Endothelial, liver, human
Species: human
Tissue Origin: liver
Kupffer Cell, human
Species: human
Tissue Origin: liver

PHH were obtained from Lonza (Walkersville, MD, USA), and HSCs, KCs and LECs were provided by Samsara (San Diego, CA, USA). PHH, HSCs and LECs were seeded in red phenolfree William E medium (A12176; Gibco, Invitrogen Corp., Waltham, MA, USA) supplemented with 5% fetal bovine serum, primary hepatocyte thawing and plating supplements solution (CM3000; ThermoFisher Scientific, Waltham, MA, USA) and 1% of a non-essential amino acids solution (11140050; Gibco).
PHHs, LECs and hepatic stellate primary cells were embedded at 0.5×106, 0.1×106 and  0.1×106 cells/mL, respectively, in a half RAFTTM 3D collagen hydrogel (016-0R92; Lonza) in 96-well plates, as recommended by the provider. Cells were cocultured in DMEM (low-glucose, pyruvate, no glutamine, no red phenol; 11054; Gibco) supplemented with bovine serum/ free fatty acid-free 0.125% solution (A7030; Sigma-Aldrich, St Louis, MO, USA), 50 U/mL penicillin/streptomycin (15140122; Gibco), dexamethasone 0.1 mM, ITS-G 1X (41400045; Gibco), GlutaMAXTM 1X (35050061; Gibco), HEPES 15 mM (15630080; Gibco), non-essential amino acids solution 1X (11140050; Gibco), acid L-ascorbic 2.5 mg/mL (A4403; Sigma-Aldrich) and glucagon 0.1 mg/mL (G2044; Sigma-Aldrich). This liver coculture medium is named “healthy media”. After 3 days of culture, the cocultures were incubated either in healthy media or in a media mimicking the NASH environment and supplemented with glucose 25 mM (Sigma-Aldrich), oleate acid 40 mM (SigmaAldrich), palmitate acid at 60 mM (Cayman Chemical, Ann Arbor, MI, USA) and tumor necrosis factor-a 5 ng/mL (PeproTech, Cranbury, NJ, USA). Healthy and NASH medium were changed every 2-3 days. At day 6, KCs were added to the coculture at 0.2×106 cells/mL. Supernatants and embedded cells were sampled on days 3, 6, 8, 10, 13 and 15 for analysis.


Background and Aims: To better understand nonalcoholicsteatohepatitis (NASH) disease progression and to evaluatedrug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture andthe NASH environment. Methods: We have developed an invitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cellsand Kupffer cells embedded in a hydrogel of rat collagen on a96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-a. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro, with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets.