Pump-less, recirculating organ-on-a-chip (rOoC) platform.

We developed a novel, pump-less directional flow recirculating organ-on-a-chip (rOoC) platform that creates controlled unidirectional gravity-driven flow by a combination of a 3D-tilting system and an optimized microfluidic layout. The rOoC platform was assembled utilizing a layer-to-layer fabrication technology based on thermoplastic materials. It features two organoid compartments supported by two independent perfusion channels and separated by a hydrogel barrier. We developed a computational model to predict wall shear stress values and then measured the flow rate in the microfluidic channels with micro-Particle-Image-Velocimetry (µPIV). The suitability of the rOoC for functional culture of endothelial cells was tested using HUVECs seeded in the perfusion channels. HUVECs aligned in response to the directional flow, formed a barrier and were able to sprout into the organoid compartments. Next, we demonstrated the viability of human stem-cell derived liver organoids in the organoid compartments. Finally, we show the possibility to circulate immune cells in the microfluidic channels that retain viability without being trapped or activated. The rOoC platform allows growing and connecting of two or more tissue or organ representations on-chip with the possibility of applying gradients, endothelial barriers, microvasculature and circulating cells independent of external tubing and support systems.

An ex Vivo Model Enables Systematic Investigation of the Intestinal Absorption and Transcytosis of Oral Particulate Nanocarriers.

Nanoparticulate formulations are being developed toward enhancing the bioavailability of orally administrated biologics. However, the processes mediating particulate carriers' intestinal uptake and transport remains to be fully elucidated. Herein, an optical clearing-based whole tissue mount/imaging strategy was developed to enable high quality microscopic imaging of intestinal specimens. It enabled the distribution of nanoparticles within intestinal villi to be quantitatively analyzed at a cellular level. Two-hundred and fifty nm fluorescent polystyrene nanoparticles were modified with polyethylene glycol (PEG), Concanavalin A (ConA), and pectin to yield mucopenetrating, enterocyte targeting, and mucoadhesive model nanocarriers, respectively. Introducing ConA on the PEGylated nanoparticles significantly increased their uptake in the intestinal epithelium (∼4.16 fold for 200 nm nanoparticle and ∼2.88 fold for 50 nm nanoparticles at 2 h). Moreover, enterocyte targeting mediated the trans-epithelial translocation of 50 nm nanoparticles more efficiently than that of the 200 nm nanoparticles. This new approach provides an efficient methodology to obtain detailed insight into the transcytotic activity of enterocytes as well as the barrier function of the constitutive intestinal mucus. It can be applied to guide the rational design of particulate formulations for more efficient oral biologics delivery.

Capturing and Quantifying Particle Transcytosis with Microphysiological Intestine-on-Chip Models.

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.

Academic User View: Organ-on-a-Chip Technology.

Organ-on-a-Chip (OoC) systems bring together cell biology, engineering, and material science for creating systems that recapitulate the in vivo microenvironment of tissues and organs. The versatility of OoC systems enables in vitro models for studying physiological processes, drug development, and testing in both academia and industry. This paper evaluates current platforms from the academic end-user perspective, elaborating on usability, complexity, and robustness. We surveyed 187 peers in 35 countries and grouped the responses according to preliminary knowledge and the source of the OoC systems that are used. The survey clearly shows that current commercial OoC platforms provide a substantial level of robustness and usability-which is also indicated by an increasing adaptation of the pharmaceutical industry-but a lack of complexity can challenge their use as a predictive platform. Self-made systems, on the other hand, are less robust and standardized but provide the opportunity to develop customized and more complex models, which are often needed for human disease modeling. This perspective serves as a guide for researchers in the OoC field and encourages the development of next-generation OoCs.

Mechanisms of uptake and transport of particulate formulations in the small intestine.

Micro- and nano-scale particulate formulations are widely investigated towards improving the oral bioavailability of both biologics and drugs with low solubility and/or low intestinal permeability. Particulate formulations harnessing physiological intestinal transport pathways have recently yielded remarkably high oral bioavailabilities, illustrating the need for better understanding the specific pathways underpinning particle small intestinal absorption and the relative role of intestinal cells. Mechanistic knowledge has been hampered by the well acknowledged limitations of current in vitro, in vivo and ex vivo models relevant to the human intestinal physiology and the lack of standardization in studies reporting absorption data. Here we review the relevant literature and critically discusses absorption pathways with a focus on the role of specific intestinal epithelial and immune cells. We conclude that while Microfold (M) cells are a valid target for oral vaccines, enterocytes play a greater role in the systemic bioavailability of orally administrated particulate formulations, particularly within the sub-micron size range. We also comment on less-reported mechanisms such as paracellular permeability of particles, persorption due to cell damage and uptake by migratory immune cells.

Hele Shaw microfluidic device: A new tool for systematic investigation into the effect of the fluid shear stress for organs-on-chips.

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.

Unlocking the Potential of Organ-on-Chip Models through Pumpless and Tubeless Microfluidics.

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.

A systematic investigation of the effect of the fluid shear stress on Caco-2 cells towards the optimization of epithelial organ-on-chip models.

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 for a 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.

Uptake of silica particulate drug carriers in an intestine-on-a-chip: towards a better in vitro model of nanoparticulate carrier and mucus interactions.

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.

Identifying human and murine M cells in vitro.

IMPACT STATEMENT: The 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.

Intestine-on-a-Chip Microfluidic Model for Efficient in Vitro Screening of Oral Chemotherapeutic Uptake.

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.