• Liver fibrosis
• collagen
• 3D models

The liver is the primary gland involved in the metabolism of xenobiotics, drugs and toxic and waste substances. It has a high regenerative capacity, due to the proliferation and differentiation of liver cells that restore the organ function. However, when the liver damage is severe and prolonged, due to
the onset of chronic liver diseases, the infiammatory response and the repair mechanisms that are activated lead to a progressive fibrosis. Fibrosis is a dynamic process that involves the progressive accumulation of extracellular matrix in an attempt to repair the damage caused by different kind of pathological conditions. Advanced liver fibrosis may result in cirrhosis, liver failure, and portal hypertension and can eventually lead to hepatocellular carcinoma. Fibrosis is promoted by myobroblasts, which are activated by infiammatory cytokines (such as TGF beta) and may derive from different types of hepatic cells, among which the main are stellate cells. Interestingly, it was demonstrated that the acquisition of broblast- and myobroblasts-phenotypes can arise during the complex biological process
known as epithelialmesenchymal transition (EMT). The studies focusing on the physiopathological mechanisms associated with liver damage, degeneration and fibrosis are of great interest, also to better
identify the risk factors associated with these phenomena. In this perspective, substantial efforts have been made to develop suitable in vitro and in vivo models mimicking liver fibrosis. For in vitro models, primary cultures of human hepatocytes are the reference model, but the difficult accessibility and the inter-individual variability,push the choice of immortalized hepatic cell lines. Several in vitro models
are based on two-dimensional cultures or co-cultures of different hepatic cell types; however, a more faithful reproduction of the in vivo situation can be obtained with three-dimensional models (3D) of cell cultures. Indeed, the 3D model allow to exploit the interactions of cells with the extracellular matrix, as well as with other cells. In recent years, methods have been developed for 3D culture based on the use of scaffolds of smart materials, of natural (collagen, extracellular matrix decellularized and digested ...) or synthesis origin (polyethylene glycol, PuraMatrixTM), that have the function of an extracellular matrix, capable of supporting cell growth and mimicking porosity, permeability and mechanical stability of the in vivo conditions.
The aim of this thesis was to set up a 3D model of liver brosis by developing a gel matrix that can be stiffened with biocompatible cross-linker and thus used to encapsulate hepatocytes. Recently, it was shown that the hardening of the extracellular matrix may result in mechanical activation of TGF beta, leading to activation of the EMT process, causing the expression of mesenchymal markers (vimentin), to the detriment of those epithelial (E-cadherin). Initially, suitable experimental conditions were optimized for obtaining 3D cellular cultures of human hepatoma HepG2 encapsulated into collagen gels.
Cell viability and proliferation were assessed by Live-Dead test, LDH assay and Trypan blue exclusion assay. The secretion albumin was selected as a marker of metabolic functionality. Unlike other methods reported in the literature and based on the use of compounds/conditions with unwanted or cytotoxic side effects, the stiffening of our collagen gels was obtained via a fully biocompatible cross-linker, i.e. the microbial enzyme transglutaminase (mTG), that catalyzes the formation of cross links between proteins. The micromechanical properties of gels were analysed by nanoindentation and the effects of gel stiffening on cell morphology, vitality and proliferation were evaluated. The expression of the epithelial-mesenchymal transition markers was evaluated by immunouorescence and western blot analysis.
Finally, in an effort to set up a more biomimetic model, the preparation of gels with a decellularized and subsequently digested hepatic extracellular matrix obtained from porcine liver was also taken into account. The results obtained indicate that the 3D collagen model is suitable to study the effects of gel stiffening by mTG up to 96 hours in culture. The stiffening of gels by low cytotoxic mTG activities resulted in a reduced secretion of albumin and in an increased expression of vimentin (i.e. two markers of EMT). These results suggest that also small changes in the mechanical properties of extracellular (micro)environment could be in support of a process - the EMT- which is supposed to contribute to hepatic fibrogenesis.