1/2/2024 0 Comments Overgrowth free download 2016![]() To do this, we adapted a recently described human gut-on-a-chip microfluidic device that enables human intestinal epithelial cells (Caco-2) to be cultured in the presence of physiologically relevant luminal flow and peristalsis-like mechanical deformations, which promotes formation of intestinal villi lined by all four epithelial cell lineages of the small intestine (absorptive, goblet, enteroendocrine, and Paneth) ( 12, 16). Thus, we set out to develop an experimental model that would overcome these limitations. This is a major limitation because establishment of stable symbiosis between the epithelium and resident gut microbiome as observed in the normal intestine is crucial for studying inflammatory disease initiation and progression ( 13), and rhythmical mechanical deformations driven by peristalsis are required to both maintain normal epithelial differentiation ( 14) and restrain microbial overgrowth in the intestine in vivo ( 15). Although higher levels of intestinal differentiation can be obtained using recently developed 3D organoid cultures ( 11), it is not possible to expose these cells to physiological peristalsis-like motions or living microbiome in long-term culture, because bacterial overgrowth occurs rapidly (within ∼1 d) compromising the epithelium ( 12). For example, intestinal epithelial cells cultured in Transwell plates completely fail to undergo villus differentiation, produce mucus, or form the various specialized cell types of normal intestine. These static in vitro methods, however, do not effectively recapitulate the pathophysiology of human IBD. Most models of human intestinal inflammatory diseases rely either on culturing an intestinal epithelial cell monolayer in static Transwell culture ( 9) or maintaining intact explanted human intestinal mucosa ex vivo ( 10) and then adding live microbes and immune cells to the apical (luminal) or basolateral (mucosal) sides of the cultures, respectively. In particular, given the recent recognition of the central role of the intestinal microbiome in human health and disease, including intestinal disorders ( 2), it is critical to incorporate commensal microbes into experimental models however, this has not been possible using conventional culture systems. However, it has not been possible to study the relative contributions of these different potential contributing factors to human intestinal inflammatory diseases, because it is not possible to independently control these parameters in animal studies or in vitro models. Suppression of peristalsis also has been strongly associated with intestinal pathology, inflammation ( 4, 5), and small intestinal bacterial overgrowth ( 5, 6) in patients with Crohn’s disease ( 7) and ileus ( 8). Various types of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, involve chronic inflammation of human intestine with mucosal injury and villus destruction ( 1), which is believed to be caused by complex interactions between gut microbiome (including commensal and pathogenic microbes) ( 2), intestinal mucosa, and immune components ( 3). ![]() ![]() Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation.
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