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Belgian Week of Gastroenterology 2019
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Thursday, February 21 • 14:40 - 14:50
Constructing a primary mouse 3D hepatic co-culture model that recapitulates HSC activation during fibrosis

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I. MANNAERTS (1), N. EYSACKERS (1), A. SMOUT (1), S. VERHULST (1), S. LEITE (1), T. ROOSENS (1), L. VAN GRUNSVEN (1) / [1] Vrije Universiteit Brussel (VUB), Jette, Belgium, Liver Cell Biology Research Group
Chronic liver disease is the major cause of progressive liver fibrosis which, in turn, leads to cirrhosis of the liver. One major obstacle in the development of efficient therapies is the lack of robust and representative in vitro models of liver fibrosis to aid in understanding the basic mechanisms of the disease and in the development phase of pharmaceuticals.
The aim of our work is to develop relevant in vitro liver fibrosis models, based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)–the main producer of scar tissue during fibrosis–, but requires cultures in which other liver cells (at least hepatocytes) are integrated.
We describe the generation of co-culture spheroids, using freshly isolated liver cells from mice. Hepatocytes and HSCs were isolated using percoll gradients and UFACS3 respectively. Spheroids were created and cultured in cell repellent plates and analyzed for RNA changes by qPCR and by immunohistochemistry for protein analysis. Cell viability and compound toxicity were determined by Cell Titer Glo assay.
We show that hepatocytes and HSCs are highly pure at the start of culture and maintain their cell-type specific marker expression over a 15-day culture period. During this period there is no major hepatocyte dedifferentiation or HSC activation, characteristics that cannot be obtained by regular 2D cultures. When exposed to TGFβ, paracetamol, or thioacetamide, we observe a compound-mediated HSC activation (direct or via hepatocytes) with a pattern similar to the in vivo HSC activation. Importantly, we can use pharmaceuticals with known anti-fibrotic properties, such as Valproic acid and Verteporfin, to reduce HSC activation in response to hepatocyte damage. Finally, genes that are differentially regulated between (2D) in vitro- and in vivo HSC activation, selected from in-house microarray and RNA-Seq data, show a more in vivo-like behavior upon paracetamol induced damage in spheroid co-cultured stellate cells compared to the classical 2D HSC cultures.
Our hepatocyte-stellate cell spheroids are a robust in vitro model of liver fibrosis. The hepatocytes can metabolize hepatotoxins and promote HSC activation. This model could facilitate the discovery of, or testing for, novel anti-fibrotic compounds as we have indications that our spheroids are a better representation of HSC activation in vivo compared to the more traditional culture models.


Thursday February 21, 2019 14:40 - 14:50 CET