THP-1/ Caco-2 co-culture as an in vitro model for the inflamed intestinal epithelium.

PRISCILA SCHILRREFF; LETICIA H HIGA; EDER L ROMERO; MARIA JOSE MORILLA

Buenos Aires

Congreso; ? Congreso III Latinoamericano de Métodos Alternativos al uso de animales; 2018

COLAMA AACYTAL

THP-1/ CACO-2 CO-CULTURE AS AN IN VITRO MODEL FOR THE INFLAMED INTESTINAL EPITHELIUMHiga LH1, Schilrreff P1 Romero EL1, Morilla MJ1 1Nanomedicine Research Program, Universidad Nacional de Quilmes, Buenos Aires, Argentinapschilrreff@unq.edu.arInflammatory bowel diseases (IBD), such as Crohn?s disease or colitis, have been postulated as being associated with both defects in the intestinal barrier and an impaired immune function. Development of novel drugs for the treatment of IBD is largely linked to animal models. However, the main problems of animal models lie in the species differences compared to human, which often causes misleading results. On the other hand, in vitro methods such as Caco-2 cell line monolayers are widely accepted as a model of the normal, healthy intestinal epithelium. Nevertheless models which consist only of enterocytes cannot mimic the complex interactions with the immune system. Thus in vitro models that adequately reproduce both healthy and inflamed intestinal tissue could provide a useful for investigating new therapeutic drugs.In this work, we describe the development and characterization of a co-culture model consisting of human intestinal cell monolayers, Caco-2 cells and THP-1 human monocyte-derived macrophages that mimics the intestine in healthy and controlled inflamed states. Caco-2 cells were cultivated for 21 days to allow intact monolayer formation. The barrier formation was monitored by Trans-Epithelial Electrical Resistance measurement (TEER) during the course of cultivation. In homoeostatic conditions without stimulation, Caco-2 and THP-1 cells were co-cultured for 24 h without affecting the barrier integrity and with no increase in the release of cytokines or ROS production. To simulate the inflamed intestine, THP-1 cells were pre-stimulated with 10 µg/ml LPS. In these conditions, Caco-2 cells showed an increase of pro-inflammatory cytokines TNF and IL-8 and ROS production. Furthermore, LPS-stimulated cells decreased TEER to 80-90% of the control value. Transmission electron microscopy of 21-day-old Caco-2 monolayers showed a continuous differentiated cell monolayer with a row of straight dense microvilli on their apical surface, interdigitations and desmosomes. The inflamed model shows a decrease in microvillus density and number of tight junctions.Immunofluorescence microscopy of Caco-2 cells was performed. Cells were fixed and stained with specific antibodies for phalloidin and occludine. In LPS stimulated cells, phalloidin (actin filaments) showed less intensity on the side which is an indicative of an reorganization process of the tight junction proteins.We therefore conclude that the co-cultivation of LPS stimulated macrophages together with the epithelial cells drives the differentiation of Caco-2 cells towards a more inflamed-like intestine characteristic.Key words: co-culture, intestine, inflammation, inflammatory bowel disease, macrophages Área temática de interes: Investigación y Desarrollo