A millifluidic human gut model with peristalsis orchestrates villus morphogenesis

The gut’s biomechanical cues are crucial features to consider when engineering the human gut and intestinal epithelial barrier in vitro. Biomimicry, however, remains an open challenge, as existing models oftentimes fail to comprehensively reproduce the kinematics of gut peristalsis, resulting in suboptimal recapitulation of intestinal function, which may have limited our knowledge of its deep implications in intestinal pathophysiology. To overcome this bottleneck, we developed a millifluidic human gut model (hGutMo) that reproduces the gut’s environment featuring physiologically relevant mechanical stimuli, including peristaltic motion and fluidic shear stress. The hGutMo uses a gelatin electrospun scaffold to support the long-term dynamic in vitro culture of human Caco-2 cells and continuously applies a relevant peristaltic stimulation and fluid flow to the cultured sample. These dynamic conditions promote extended cell viability for 28 days of continuous dynamic culture, as well as the spontaneous morphogenesis of three-dimensional villus-like structures, closely resembling those in the human small intestine, as early as 7 days of culture. The Caco-2 cells lining these structures express villin and form adherens junctions, indicating epithelial cohesion. Moreover, the villus-like protrusions significantly increase the surface area compared to Caco-2 monolayers. The ability of the hGutMo system to recapitulate key biomechanical and structural features of the human gut makes it a promising in vitro model that could be useful in intestinal tissue engineering for investigating intestinal mechanobiology and uncovering mechanotransduction pathways.