citations

                    The role of sinusoidal endothelial cells and TIMP1 in the regulation of fibrosis in a novel human liver 3D NASH model
                

Authors:

van Riet, Sander; Julien, Anais; Atanasov, Andrea Nordling, Åsa; Ingelman-Sundberg, Magnus

In:

Source: Hepatology
Publication Date: (2024)
Issue: 8: e0374.

Research Area:

Gastroenterology

Toxicology

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

Experiment

Primary human hepatocytes (PHH), crude fractions of NPC, and LSECs were obtained from LONZA or KaLyCell. The donor characteristics and supporting information are presented in Table 1. Spheroids were cultured as described.[13,15] Briefly, PHH were seeded in 96-well Corning Costar Ultra-Low Attachment Plates (Merck, Kenilworth, NY) or in 96-Well Nunclon Sphera U-Shaped-Bottom Microplate plates (Thermo Fisher, Waltham, MA) in the following compositions: (i) PHH alone (monoculture), (ii) PHH/NPC (coculture), and (iii) PHH/NPC/LSEC (triple culture). For different spheroid compositions, PHH/NPC and PHH/NPC/LSEC were seeded at ratios of 1500/375 and 1500/375/200 cells per well, respectively. Spheroids were seeded in William’s E medium supplemented with 2 mM L-glutamine (SigmaAldrich, Saint Louis, MO), 100 U/mL penicillin (SigmaAldrich), 100 µg/mL streptomycin (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), ITS X-100 (Thermo Fisher), and 10% fetal bovine serum (FBS; Thermo Fisher).

Abstract

Background:

The prevalence of NAFLD is rapidly increasing. NAFLD can progress to NASH, fibrosis, cirrhosis, and HCC, which will soon become the main causes of liver transplantation. To date, no effective drug for NASH has been approved by the Food and Drug Administration. This is partly due to the lack of reliable human in vitro models. Here, we present a novel human liver spheroid model that can be used to study the mechanisms underlying liver fibrosis formation and degradation.

Methods and Results:

Such spheroids, which contain hepatocytes, stellate cells, KC, and LSECs, spontaneously develop fibrosis that is exacerbated by treatment with free fatty acids. Conditioned medium from activated LSECs caused similar activation of fibrosis in spheroids containing primary human hepatocyte and NPCs, indicating the action of soluble mediators from the LSECs. Spheroids containing LSECs treated with free fatty acids produced tissue inhibitor of metalloproteinases inhibitor 1, a matrix metalloproteinases inhibitor important for fibrosis progression. Tissue inhibitor of metalloproteinases inhibitor 1 knockdown using siRNA led to a reduction in collagen and procollagen accumulation, which could be partially rescued using a potent matrix metalloproteinases inhibitor. Interestingly, tissue inhibitor of metalloproteinases inhibitor 1 was found to be expressed at higher levels, specifically in a subtype of endothelial cells in the pericentral region of human fibrotic livers, than in control livers.

Conclusion:

Potential anti-NASH drugs and compounds were evaluated for their efficacy in reducing collagen accumulation, and we found differences in specificity between spheroids with and without LSECs. This new human NASH model may reveal novel mechanisms for the regulation of liver fibrosis and provide a more appropriate model for screening drugs against NASH.

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