Long-term culturing of Giardia trophozoites with human epithelial cells in vitro is challenging due to the microaerophilic nature of the parasite. To overcome the parasite survival barrier, most in vitro studies have utilized high multiplicities of infection in addition to the short incubation times. These conditions do not reflect a typical Giardia infection where a low infectious dose leads to an active infection that can span several days to weeks. Therefore, establishing an in vitro model that allows for the protracted coculture of host epithelial and immune cells with Giardia trophozoites would greatly contribute to the understanding of late infection interactions. Our model utilizes transwell inserts to co-culture a human intestinal epithelial cell line and a 
murine peritoneal macrophage cell line in a manner that represents the apical-basolateral orientation of the small intestine. IC21 murine peritoneal macrophages were selected for their similarity to human macrophages, including their typical macrophage morphology in culture, their expression of macrophagespecific antigens, their activation by lipopolysaccharide, their phagocytic ability, and their expression of IgG receptors. Caco-2 cells, derived from a human colonic adenocarcinoma retain both morphologic and phenotypic characteristics of intestinal epithelial cells when fully differentiated, including polarized morphology, microvilli on the apical surface, expression of brush border enzymes, and adjacent cell tight junctions. The in vitro Albaspidin-AA differentiation of Caco-2 cells into a phenotype similar to small intestinal epithelial cells is a timemediated event that is Alprostadil dependent on many factors, including passage number, seeding density, media composition, and substrate support. In our study, the use of Caco-2 cells at three days post plating allows for the assessment of how the parasite affects the proliferation and differentiation process of enterocytes in the intestine. The epithelial barrier of the intestine is replenished every 4�C5 days; therefore, enterocyte renewal through stem cell differentiation is critical for normal functioning of the human gut. Caco-2 cells have been used to model the differentiation process of enterocytes in the small intestine. Although immature proliferating Caco2 cells show differences in gene expression, the protein expression profiles are remarkably similar when compared to fully differentiated Caco-2 cells. Thus far, Caco-2 cells are the best described enterocyte cell line and the most common epithelial cell line used in in vitro Giardia�Chost interactions. Therefore, using Caco-2 cells to characterize our co-culture model allows us to compare our results with those published in the literature. Following establishment of cell-cell communication and epithelial monolayer formation, Giardia trophozoites were added to the system using a lower starting density than what has previously been reported. This model allows the parasite to proliferate in culture, more accurately reflects the infection in vivo, and allows for the characterization of host-Giardia interactions from the start of an infection through its termination, including the role of immune cells in limiting the infection. Our data indicate that both parasites and epithelial cells are viable in the 90% DMEM/10% Giardia media mixture. Using transwell inserts filled with medium, we were able to limit the oxygen exposure of the parasites, while allowing the epithelial cells to exchange oxygen and nutrients through their basolateral surface. Giardia proliferates in the 90% DMEM/10% Giardia media and saturates the insert surface at 5 days post-infection. The drop in parasite density after 5 days is attributed to daily feeding of the insert cultures.