Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis

PLoS One. 2017 Jun 30;12(6):e0179995. doi: 10.1371/journal.pone.0179995. eCollection 2017.


Background & aims: Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA).

Methods: Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated.

Results: We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed.

Conclusion: Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.

MeSH terms

  • Cell Line
  • Extracellular Matrix / metabolism*
  • Hepatic Stellate Cells / drug effects*
  • Hepatic Stellate Cells / metabolism
  • Hepatic Stellate Cells / pathology
  • Humans
  • Lipopolysaccharides / pharmacology
  • Liver Cirrhosis / metabolism*
  • Liver Cirrhosis / pathology
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Methotrexate / pharmacology
  • NF-E2-Related Factor 2 / metabolism*
  • Thioacetamide / pharmacology
  • Transforming Growth Factor beta1 / pharmacology
  • Tumor Necrosis Factor-alpha / pharmacology


  • Lipopolysaccharides
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • Transforming Growth Factor beta1
  • Tumor Necrosis Factor-alpha
  • Thioacetamide
  • Methotrexate

Grant support

The work performed at FHNW was partly funded by the CTI (Commission for Technology and Innovation) and partly by SCAHT (Swiss Center for Applied Human Toxicology), both Swiss federal organisations. The studies performed at InSphero AG were funded internally. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.