Human hepatocytes in three-dimensional culture on Innovative biopolymeric scaffolds as a useful system for in vitro toxicology tests

P 1.6 HUMAN HEPATOCY TES IN THREE- DIMENSIONAL CULTURE ON INNOVATIVE BIOPOLYMERIC SCAFFOLDS AS AN USEFUL SYSTEM FOR IN VITRO TOXICOLOGY TESTS Stampella A. (a), Massimi M. (b), Barbetta A. (c), Rizzitelli G. (c), Dentini M. (c), Conti Devirgiliis L. (a) (a) Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy (b) Department of Basic and Applied Biology, University of L’Aquila, L’Aquila, Italy (c) Department of Chemistry, Sapienza University of Rome, Rome, Italy Many innovative biomaterials have recently be developed as scaffolds to replace physiological matrix components and their im provement has led to significant advances in culture techniques in terms of cell survival, quantitative expansion, maintenance of differentiated phenotype and specific cell functions. A key point in achieving these goals has been to maintain a three-dimensional culture and the typical cyto-architecture of the tissue by improving the extracellular matrix geometry and by promoting cell-cell contacts and reciprocal adhesions. These bio-artificial systems represent a real hope as functional substitutes for damaged organs and tissues and have provoked a great interest in the field of regenerative medicine. Concerning hepatocyte cultures, since the liver is the main organ involved in detoxification processes and in the defence of organisms against harmful molecules, in addition to their biomedical applications, these systems can be utilized as invaluable tool for toxicology tests for analyzing the effects on metabolism of new drugs, or for screening potentially toxic substances. The aim of our research was to identify the most suitable biomaterial for the technological applications with hepatocytes. Since the possibility to improve the performance of thes e systems depends strongly on the methods used to create the scaffolds, here we analyzed porous matrices made of gelatin or blends of gelatin and glycosaminoglycans, obtained with different methods for the culture of the C3A cell line, considered a good model of human hepatocytes. Scaffolds were obtained using either a concentrated emulsi on-templating technique known as High Internal Phase Emulsion (HIPE) or a gas foaming technique; the latter method uses an inert gas instead of the internal liquid phase toluene, avoiding the use of organic solvent and allowing the creation of scaffolds with la rger pores and interconnections. Cell viability was analysed using MTS and LDH assays; ultrastructural morphology and three-dimensional cell organization into the scaffold were assessed by SEM; albumin and urea secretion, as the main metabolic markers of hepatocyte functions, were monitored using, respectively, an ELISA kit and a colorimetric assay. Finally Cytochrome P450-3A4 activity was quantified by a luminescent method. Values of activity of this important enzyme of the detoxification system, obtained in the absence or in the pr esence of specific inducing molecules, were compared between the different culture conditions.