ECM-BASED IN VITRO 3D-MODELS OF THE LIVER FOR HEPATOTOXICITY TESTING

In the context of drug development, liver plays a crucial role. It is not only directly responsible for drug metabolism, but it is also implied in hundreds of viable functions, whose impairment might produce differences in active principle processing, and pharmacokinetics, leading to the outbreak of severe adverse reactions (Rodighiero, 2012), including hepatoxic effects. This work will present the first developmental steps of an ECM-based, in vitro 3D-model of the liver, to assess drugs hepatotoxicity. ECM was obtained from decellularized porcine liver, by a combination of different methods (Mazza et al., 2017). The decellularization buffer was injected in multiple sites of 0.5 cm cubes of liver and then used to incubate the samples while under orbital stirring up to 7 days. Lyophilized cubes were grinded after freezing in liquid N2. ECM powder (1.4% w/v) was added in an alginate (ALG) solution (3.5% w/v) in complete medium. The hydrogel was characterized by rheological testing and the stability in medium was evaluated up to 14 days. For cell loaded hydrogels, HepG2 cells were suspended in the ALG-ECM suspension (2x106 cell/ml) prior crosslinking (Lan et al., 2010). MTT test and confocal microscopy, with live/dead kit were employed to evaluate viability and spatial distribution. The produced hydrogel shows rheological characteristics reproducing the ones of the liver tissue and is stable up to 11 days. Loaded HepG2 cells were viable and homogeneously distributed within the 3D-matrix. Their number increased upon time and the production of physiological-like aggregates is observed. The model is now being employed for the study of hepatoxicity to the administration of various drugs (i.e. acetaminophen, midazolam, chlordiazepoxide). Furthermore, the developed model can be tailored to mimic not only physiological organs, but also diseased ones; this study is integrated in the development of new approach methodologies applied to toxicology and preclinical drug evaluation.