Sviluppo di una co-coltura come modello della barriera intestinale utilizzando cellule epiteliali Caco-2 ed HT29-MTX

The small intestine is the most important site of the gastrointestinal tract for the absorption of orally delivered drugs. It owns a characteristic conformation represented by the villi, finger-like formations, which greatly increase the surface of absorption. The main kinds of intestinal cells are enterocytes, goblet-like cells, entero-endocrine cells, Paneth cells and M cells. Several models of intestinal barrier have been developed to investigate and predict drug absorption in human gut. They can be classified into in vivo, in situ, in vitro cell-free and in vitro cell-based models. This project focuses over in vitro cell-based models and in particular over co-cultures made up of Caco-2 and HT29-MTX epithelial cells, originating both from a colorectal adenocarcinoma in the 1970s by Dr. Fogh. Generally Caco-2 cells are grown in mono-cultures and results obtained from permeability studies show a good correlation with human values. However this kind of mono-culture lacks some important characteristics of human gut: first of all the production and presence of a mucus layer. Besides in Caco-2 cells paracellular permeability is far lower, due to the numerous and highly sealed tight junctions, transepithelial resistance (TEER) is higher and P-glycoprotein is over-expressed resulting in an increased basolateral to apical efflux [1]. Therefore to overcome these limitations and predict drug transport and absorption in a more accurate way, Caco-2 cells were co-cultured with HT29-MTX cells, which are goblet-like cells. HT29-MTX produce mucus, do not express P-glycoprotein, have leakier tight junctions and hence higher paracellular permeability and lower values of TEER, by comparison with Caco-2 cells. The aim of this study was to obtain a co-culture owning features closer to human intestine. In the development of a co-culture many factors could be changed, such as the culture medium, the seeding density and the Caco-2/HT29-MTX cells ratio [2]. Within this work one crucial parameter was demonstrated to strongly influence the properties of the co-culture. The results of this work, carried out within the LLP/Erasmus project in collaboration with the department of Galenic Pharmacy at the University of Franche-Comté, have been covered by an international patent.