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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.

M cells are an epithelial cell population found in the follicle-associated epithelium overlying gut-associated lymphoid tissues. They are specialized in the transcytosis of luminal antigens. Their transcytotic capacity and location in an immunocompetent environment has prompted the study of these cells as possible targets for oral drug delivery systems. Currently, the models most commonly used to study M cells are restricted to in vivo experiments conducted in mice, and in vitro studies conducted in models comprised either of primary epithelial cells or established cell lines of murine or human origin. In vitro models of the follicle-associated epithelium can be constructed in several ways. Small intestinal Lgr5+ stem cells can be cultured into a 3D organoid structure where M cells are induced with RANKL administration. Additionally, in vitro models containing an “M cell-like” population can be obtained through co-culturing intestinal epithelial cells with cells of lymphocytic origin to induce the M cell phenotype. The evaluation of the efficiency of the variations of these models and their relevance to the in vivo human system is hampered by the lack of a universal M cell marker. This issue has also hindered the advancement of M cell-specific targeting approaches aimed at improving the bioavailability of orally administered compounds. This critical review discusses the different approaches utilized in the literature to identify M cells, their efficiency, reliability and relevance, in the context of commonly used models of the follicle-associated epithelium. The outcome of this review is a clearly defined and universally recognized criteria for the assessment of the relevance of models of the follicle-associated models currently used.Impact statementThe study of M cells, a specialized epithelial cell type found in the follicle-associated epithelium, is hampered by the lack of a universal M cell marker. As such, many studies lack reliable and universally recognized methods to identify M cells in their proposed models. As a result of this it is difficult to ascertain whether the effects observed are due to the presence of M cells or an unaccounted variable. The outcome of this review is the thorough evaluation of the many M cell markers that have been used in the literature thus far and a proposed criterion for the identification of M cells for future publications. This will hopefully lead to an improvement in the quality of future publications in this field.

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

Many highly effective chemotherapeutic agents can only be administered intravenously as their oral delivery is compromised by low gastro-intestinal solubility and permeability. SN-38 (7-ethyl-10-hydroxycamptothecin) is one such drug; however, recently synthesized lipophilic prodrugs offer a potential solution to the low oral bioavailability issue. Here we introduce a microfluidic-based intestine-on-A-chip (IOAC) model, which has the potential to provide new insight into the structure-permeability relationship for lipophilic prodrugs. More specifically, the IOAC model utilizes external mechanical cues that induce specific differentiation of an epithelial cell monolayer to provide a barrier function that exhibits an undulating morphology with microvilli expression on the cell surface; this is more biologically relevant than conventional Caco-2 Transwell models. IOAC permeability data for SN38 modified with fatty acid esters of different chain lengths and at different molecular positions correlate excellently with water-lipid partitioning data and have the potential to significantly advance their preclinical development. In addition to advancing mechanistic insight into the permeability of many challenging drug candidates, we envisage the IOAC model to also be applicable to nanoparticle and biological entities. Refereed/Peer-reviewed

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