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3D co-culture of fatty hepatocytes and stellate cells: evaluation of fibrogenesis induced by steatosis in a new in vitro model of nonalcoholic fatty liver disease

Abstract

The nonalcoholic fatty liver disease (NAFLD) is closely related to obesity and can evolve from steatosis to steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. Steatosis is considered an important risk factor in the progression of fibrosis, mostly by the secretion of cytokines and growth factors by the fatty hepatocytes. The paracrine release of these substances promotes the activation and the proliferation of hepatic stellate cells (HSCs), the main cells involved in liver fibrogenesis. In vitro models can simulate some physiopathogenic aspects of the NAFLD, reducing financial costs and the use of experimentation animals in the pre-clinical tests as well as accelerating the screening process of new molecules. However, the traditional monolayer cell culture (2D) presents some physiological limitations, and do not allow direct interaction between different cellular types in co-culture systems. The 3D culture of multicellular spheroid clusters proposed in this project aims to mimic the microenvironment that can be found in the liver, allowing the interaction between cells and their byproducts in co-culture models. Thus, this work aims to establish and validate an unpublished model of 3D cell culture of the NAFLD, and evaluate the fibrogenesis mechanisms induced by steatosis in this in vitro model. The 3D co-culture model will be obtained by the co-culture of lineages of hepatocytes C3A/HepG2 (that will be induced to steatosis by the incubation of free fatty acids) and LX-2 human hepatic stellate cells. After the culture of spheroids for 24, 48 and 72 hours, analysis of viability and cellular cytotoxicity will be analyzed. The spheroids will be also histologically processed for the histochemical analysis and quantification of fibrosis and lipid vacuoles. The morphologic pattern of cell distribution in the spheroids will be evaluated with immunofluorescence, confocal microscopy and transmission electron microscopy. Also, pro-inflammatory and pro-fibrogenic cytokines secreted by cellular spheroids will be dosed, as the type I collagen produced by HSCs. The molecular mechanisms of hepatic lipogenesis and fibrogenesis will be analyzed by the gene expression of several molecules involved in these processes. This in vitro model will allow faster progress in drug testing, and help understand the mechanisms involved in the transition between fatty liver disease and NASH, assisting in the development of new therapeutic strategies. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
ROMUALDO, GUILHERME RIBEIRO; DA SILVA, TEREZA CRISTINA; DE ALBUQUERQUE LANDI, MARINA FROTA; MORAIS, JULIANA AVILA; BARBISAN, LUIS FERNANDO; VINKEN, MATHIEU; OLIVEIRA, CLAUDIA PINTO; COGLIATI, BRUNO. Sorafenib reduces steatosis-induced fibrogenesis in a human3Dco-culture model of non-alcoholic fatty liver disease. ENVIRONMENTAL TOXICOLOGY, v. 36, n. 2 SEP 2020. Web of Science Citations: 0.

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