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Refereed/Peer-reviewed Understanding the intestinal transport of particles is critical in several fields ranging from optimizing drug delivery systems to capturing health risks from the increased presence of nano- and micro-sized particles in human environment. While Caco-2 cell monolayers grown on permeable supports are the traditional in vitro model used to probe intestinal absorption of dissolved molecules, they fail to recapitulate the transcytotic activity of polarized enterocytes. Here, an intestine-on-chip model is combined with in silico modeling to demonstrate that the rate of particle transcytosis is ≈350× higher across Caco-2 cell monolayers exposed to fluid shear stress compared to Caco-2 cells in standard “static” configuration. This relates to profound phenotypical alterations and highly polarized state of cells grown under mechanical stimulation and it is shown that transcytosis in the microphysiological model is energy-dependent and involves both clathrin and macropinocytosis mediated endocytic pathways. Finally, it is demonstrated that the increased rate of transcytosis through cells exposed to flow is explained by a higher rate of internal particle transport (i.e., vesicular cellular trafficking and basolateral exocytosis), rather than a change in apical uptake (i.e., binding and endocytosis). Taken together, the findings have important implications for addressing research questions concerning intestinal transport of engineered and environmental particles.

This method describes a novel approach to systematically investigate the effect of the fluid shear stress (FSS) on epithelial cells thanks to a single microfluidic device based on Hele-Shaw geometry. The method was validated with intestinal Caco-2 cell monolayers and lung A549 cells. We provide guidelines to adjust the experimental parameters to apply specific ranges of FSS and to specify more accurately the area where to image the cells within the device by the performance of a computational simulation of the fluid flow. Most importantly, this simulation enables to validate the equation. This approach was successfully applied to systematically investigate Caco-2 cell monolayers-based intestine-on-chip models as reported in a companion article published in Biomaterials. This study showed that exposure to microfluidic FSS induces significant phenotypical and functional changes. A detailed understanding of the effects of the FSS will enable the realization of in vitro organs-on-chip models with well-defined characteristics tailored to a specific purpose. The Hele-Shaw approach used in this study could be readily applied to other cell types and adapted for a wide range of physiologically relevant FSS.•Fluid shear stress is a key parameter in the differentiation of epithelial cells cultured in organ-on-chip models.•A simple approach can be used to assess the effect of fluid shear on cellular monolayer cultured in microfluidic devices.•Careful optimization of fluid shear stress environment is necessary for the development of better-defined organ-on-chip models.•Computational simulation of the fluid flow gives an accurate definition of the FSS in a microfluidic channel necessary to interpret the results., Graphical abstract Image, graphical abstract

Micro and nano-particulate carriers have potential to increase bioavailability of oral drugs, but must first overcome the mucus barrier of the intestinal epithelium to facilitate absorption and entry to systemic circulation. We report on mucus-silica nanoparticulate carrier interactions in an in vitro intestine-on-a-chip (IOAC) microfluidic model. Caco-2 cells cultured within the IOAC model recapitulate the morphology of the human intestinal epithelium that is currently lacking in traditional static Transwell models. Fine control over the cell culture conditions produced a mucus layer, previously problematic to achieve without employing cell co-culture. The microdevice design also allowed for direct imaging of silica particulate carrier (40–700 nm) uptake through the mucus and cellular monolayer. PEGylated particulate carriers penetrated more readily through the mucus layer compared to non-PEGylated particulate carriers while larger particulate carriers tended to retard particulate carrier penetration through a dense mucus mesh. This was confirmed via imaging flow cytometry and UV-fluorescence spectroscopy. The IOAC also demonstrated the ability to mimic intestinal peristaltic fluidic conditions, which in turn affects the particulate carrier uptake. This in vitro IOAC model has potential to directly elucidate mucus interactions and uptake mechanisms for a range of drug carrier systems usc Refereed/Peer-reviewed

Microfluidic organs‐on‐chips are rapidly being developed toward eliminating the shortcomings of static in vitro models and better addressing basic and translational research questions. A critical aspect is the dynamic culture environment they provide. However, the associated inherent requirement for controlled fluid shear stress (FSS) and therefore the need for precise pumps limits their implementation. To address this issue, here a novel approach to manufacture pumpless and tubeless organs‐on‐chips is reported. It relies on the use of a hydrophilic thread to provide a driving force for the perfusion of the cell culture medium through constant evaporation in the controlled conditions of a cell incubator. Well‐defined and tuneable flow rates can be applied by adjusting the length and/or diameter of the thread. This approach for the preparation of an intestine‐on‐chip model based on the Caco‐2 cell line is validated. Five days culture under 0.02 dyn·cm−2 shear conditions yield monolayers similar to those prepared using a high‐precision peristaltic pump. A pumpless device can also be used to delineate the effect of FSS on the phenotype of adenocarcinomic human alveolar basal epithelial A549 cells. It is anticipated that the pumpless approach will facilitate and herefore increase the use of organs‐on‐chips models in the future. Refereed/Peer-reviewed

Epithelial cells experience constant mechanical forces, including fluid shear stress (FSS) on their apical surface.These forces alter both structure and function. While precise recapitulation of the complex mechanobiology of organs remains challenging, better understanding of the effect of mechanical stimuli is necessary towards the development of biorelevant in vitro models. This is especially relevant to organs-on-chip models which allow for fine control of the culture environment. In this study, the effects of the FSS on Caco-2 cell monolayers were systematically determined using a microfluidic device based on Hele-Shaw geometry. This approach allowed fora physiologically relevant range of FSS (from ∼0 to 0.03 dyn/cm2) to be applied to the cells within a single device. Exposure to microfluidic FSS induced significant phenotypical and functional changes in Caco-2 cell monolayers as compared to cells grown in static conditions. The application of FSS significantly altered the production of mucus, expression of tight junctions, vacuolization, organization of cytoskeleton, formation of microvilli, mitochondrial activity and expression of cytochrome P450. In the context of the intestinal epithelium,this detailed understanding of the effects of the FSS will enable the realization of in vitro organs-on-chip models with well-defined and tailored characteristics to a specific purpose, including for drug and nanoparticle absorption studies. The Hele-Shaw approach used in this study could be readily applied to other cell types and adapted for a wide range of physiologically relevant FSS. Refereed/Peer-reviewed