Authors E. VAN OS (1), L. COOLS (1), N. EYSACKERS (1), A. SMOUT (1), S. VERHULST (1), H. Reynaert (1), I. MANNAERTS (1), L. VAN GRUNSVEN (1) / [1] Vrije Universiteit Brussel (VUB), Jette, Belgium, BMWE-LIVR
Introduction Sustained liver disease independent of the etiology leads to liver fibrosis. Drug induced liver injury (DILI), metabolic (dysfunction) associated fatty liver disease (MAFLD), fibrosis and subsequently cirrhosis are a great health burden and a major cause of death worldwide, for which currently no therapies are available. During chronic liver disease hepatic stellate cells (HSCs) become activated and will obtain a myofibroblast phenotype and produce extracellular matrix that accumulates in the liver giving rise to scar tissue. However, not only HSCs but also other non-parenchymal cells respond to liver injury such as liver sinusoidal endothelial cells(LSECs) and Kupffer cells(KCs). These cells produce molecules that can lead to and aggravate HSCs activation and fibrosis. Currently, there are no robust in vitro culture models available that can recapitulate the complex fibrotic response upon hepatocyte damage or MAFLD, and thus animal studies remain necessary to test and screen drugs.
Aim Our aim was to develop a multicellular spheroid in vitro model with primary isolated mouse HSCs, KCs, LSECs and hepatocytes that can be used to model DILI, fibrosis and MAFLD and for research into its mechanism of action and the development of new anti-fibrotic/MAFLD therapies.
Methods Multicellular liver spheroids were generated by the co-culture of freshly isolated primary mouse hepatocytes, isolated by Percoll gradient isolation, and LSECs, HSCs and KCs isolated by FACS. DILI was induced by 24h exposure of DILI compounds and toxicity was determined by an ATP cell viability assay. Chronic injury was induced by 72h exposure with Acetaminophen(APAP) and MAFLD induction was obtained by culturing spheroids in lipogenic media (oleic and palmitic acid) for 9 days. Furthermore, treatment of the fibrotic response in both chronic liver injuries was done by addition of PPAR agonists Pioglitazone, Elafibranor and Lanifibranor to the medium. Intracellular fat accumulation in the spheroids was assessed by bodipy staining, mRNA expression monitored by RT-qPCR and protein changes detected with immunohistochemistry and Enzyme-Linked Immuno Sorbent Assay.
Results We established 3D co-cultures with freshly isolated liver cells from mice that can be cultured for at least 14 days without significant change in cellular composition or induction of HSC activation. Stellate cells could be activated directly by exposure to TGFβ as shown by a significant upregulation of HSC markers like Acta2 and Col1a1 at the mRNA level and as well as Collagen protein levels determined by ELISA. DILI was induced by an acute or chronic exposure of APAP and chronic exposure of APAP generated a fibrotic response with an upregulation of Acta2 and Collagen genes, specific for HSC activation, as well as collagen deposition in the spheroids and secretion of the protein in the culture media. Exposure of spheroids to high amounts of free fatty acids resulted in MAFLD induction evidenced by an intracellular lipid accumulation and change in pro-fibrotic (Acta2, Col1a1, Col3a1, Col5a2) and inflammatory genes (Il-1 and Il-6). Interestingly, preliminary data indicates that the fibrotic response in APAP-exposed spheroids could be inhibited by PPAR agonists and anti-fibrotic/MAFLD drug candidates Pioglitazone, Elafibranor and Lanifibranor.
Conclusions We established a robust multicellular in vitro spheroid culture model from primary mouse liver cells that can display a fibrotic response upon chronic exposure to APAP or under MAFLD conditions. Moreover, this model faithfully represents characteristics of MAFLD. In addition, these primary liver spheroids can potentially be used to assess novel anti-fibrotic/MAFLD compounds and for the development of novel therapies. *two first authors contributed equally **last two authors contributed equally
