DEVELOPMENT OF A CACO2/HT-29 70/30 CO-CULTURE AS AN IN VITRO MODEL OF HEALTHY OR OBESE HUMAN INTESTINE AND ITS VALIDATION FOR STUDYING NUTRIENT/INTESTINE INTERACTIONS AT MOLECULAR AND BIOCHEMICAL LEVEL

The Ph.D. project has been structured in three different parts: The first one focused on setting up and characterizing a 70/30 Caco2/HT-29 co-culture as an in vitro model to mimic the physiology of the human intestinal epithelium starting from the two parental cell populations already differentiated. The co-culture morpho-functional features were analyzed at confluence (T0) and 3, 6, 10 and 14 days after T0 (T3, T6, T10 and T14, respectively). Morphological analysis revealed: at T6, the presence of microvilli, a complete paracellular junctional apparatus and mucus; at T14, abundant microvilli and mucus absence. The functional analysis showed: an increase of Alkaline Phosphatase, Aminopeptidase N and Dipeptidyl Peptidase IV specific activity with days in culture, indicative of a progressive acquisition of a differentiated intestinal phenotype; permeability values comparable to the ones of the human small intestine and indicative of a good status of the tight junctions. This co-culture could be considered a more versatile, suitable and simpler in vitro model of human small intestinal epithelium, than the previous published ones, able to reach the intestinal differentiated phenotype already after six days from the confluence. The second part focused on the validation of the 70/30 Caco2/HT-29 co-culture as model to study absorption and nutrient/food-intestine interactions at molecular and biochemical level. Five different experimental water biscuits and breads were subjected to an in vitro gastrointestinal digestion and the obtained digestates were administered to the co-culture at physiological doses, calculated starting from the daily recommended dose in relation to the total surface area of intestinal absorption, and in excess. The effect of digestates administration on co-culture cell viability, paracellular permeability, oxidative status and anorectic hormones production were evaluated: all the digestates were not able to affect co-culture viability or to modify the paracellular permeability; all the water biscuit digestates were able to reduce intracellular ROS formation due to the natural presence of antioxidant compounds in the flours used to prepare them; all the bread samples were able to modulate the co-culture anorectic hormone production. These studies showed the suitability of the co-culture to study a wide range of interactions between nutrients/food/nutraceuticals and intestine. The third part focused on setting up and characterizing an in vitro cellular model of obese/overweight intestine using the 70/30 Caco2/HT-29 co-culture. The nutrient excess was mimed increasing the frequency of medium change compared to the standard condition and the co-culture morpho-functional features were analyzed at 3, 7, 11 and 15 days after confluence (T3, T7, T11 and T15, respectively). The obtained in vitro model, after 15 days of post-confluence and compared to our standard co-culture, showed a higher: i) inflammatory and oxidative status; ii) paracellular permeability; iii) microvilli enzyme activity; iv) production of anorectic hormones. These features were comparable to the ones revealed in vivo in the intestine of obese subjects, thus validating the present cellular model to study obesity-associated modifications at the molecular level.