Molecular aspects of cellular spheroids in an in vitro 3D model of nonalcoholic fatty liver disease (NAFLD)

Abstract

Molecular aspects of cellular spheroids in an in vitro 3D model of nonalcoholic fatty liver disease ABSTRACTThe progressive growth of obesity in human population has contributed to increase the occurrence of nonalcoholic fatty liver disease (NAFLD). NAFLD can be defined as the excessive accumulation of lipids in hepatocytes, causing steatosis, which can progress to steatohepatitis, fibrosis, cirrohosis and hepatocellular carcinoma. In steatosis, fatty liver cells secrete cytokines and growth factors that promote activation and proliferation of stellate cells (CEHs), which are responsible for liver fibrogenesis. Nowadays, the only available and effective treatment for end-stage liver disease is transplantation, showing the necessity of the development of new drugs and therapies that contribute to improve patient survival. The experimental models in vitro monolayer (2D) are widely used to simulate certain aspects of NAFLD facilitating the development of new molecules and reducing the use of experimental animals in preclinical testing. However, these models show physiological limitations, considering interaction of different cell types in coculture systems, furthermore, monolayer model does not mimic the three-dimensional environment liver. Moreover, the cultivation 3D model seems to be a better option to simulate the liver microenvironment and allow interaction between cells and their byproducts. In this context, this paper aims to establish and validate a new model of 3D coculture of hepatocytes induced steatosis and hepatic stellate cells (CEHs), inducing the formation of cellular spheroids. The induction of steatosis will mimic NAFLD and will be achieved by coculture of strains C3A/HepG2 hepatocytes incubated with non-esterified fatty acids and stellate cells LX-2 human. The 3D model is obtained by coculture cell in plates that allow low adherence, favoring the formation of multicellular spheroids. After culturing the spheroids during 24, 48 and 72 hours cytotoxicity,viability of cells, and the dosage of triglycerides intracellular will be dosed. Proinflammatory and profibrogenic cytokines secreted by spheroids and collagen type I produced by CEHs will also be dosed. The development of hepatic microenvironment in 3D coculture will allow rapid progress in the development of new therapeutic strategies in NAFLD and reduction on the number of animals used in experimentation , also to a better understanding of the molecular mechanisms that promote the development of NAFLD to fibrosis. (AU)