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Morphologic and ultrastructural evaluation of cellular spheroids in an in vitro 3D model of nonalcoholic fatty liver disease

Grant number: 11/18461-9
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): January 01, 2012
Effective date (End): December 31, 2012
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Bruno Cogliati
Grantee:Marina Frota de Albuquerque Landi
Home Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The nonalcoholic fatty liver disease (NAFLD) is closely related to obesity and can be described as the excessive accumulation of triglycerides in the hepatocytes, with the possibility of evolving from steatosis to steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. Currently, nonalcoholic steatohepatitis (NASH) is the third leading cause of liver transplant indication. Therefore, it is necessary to develop new drugs and more effective therapies. In vitro models can simulate some physiopathogenic aspects of the NAFLD, reducing financial costs and the use of experimentation animals at the pre-clinical tests as well as accelerating the development of new molecules. However, the traditional monolayer cell culture presents some physiological limitations that can be overcome by the three-dimensional model proposed in this project. The 3D culture of multicellular spheroid clusters 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 Thus, this work aims to establish and validate an unpublished model of 3D cell culture of the NAFLD, which will be obtained by the co-culture of lineages of hepatocytes C3A/HepG2 (that will be induced to steatosis by the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 stellate cells. The 3D co-culture model will be obtained by the cell culture in plates with low adherence that allows the formation of multicellular spheroids. After the culture of spheroids for 24, 48 and 72 hours, the cytotoxicity and the intracellular triglycerides content 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 and quantification of activated stellate cells and hepatocytes. 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)