Effects of allopurinol and febuxostat on uric acid transport and transporter expression in human umbilical vein endothelial cells.

Uric acid induces radical oxygen species formation, endothelial inflammation, and endothelial dysfunction which contributes to the progression of atherosclerosis. Febuxostat inhibits BCRP- and allopurinol stimulates MRP4-mediated uric acid efflux in human embryonic kidney cells. We hypothesized that endothelial cells express uric acid transporters that regulate intracellular uric acid concentration and that modulation of these transporters by febuxostat and allopurinol contributes to their different impact on cardiovascular mortality. The aim of this study was to explore a potential difference between the effect of febuxostat and allopurinol on uric acid uptake by human umbilical vein endothelial cells. Febuxostat increased intracellular uric acid concentrations compared with control. In contrast, allopurinol did not affect intracellular uric acid concentration. In line with this observation, febuxostat increased mRNA expression of GLUT9 and reduced MRP4 expression, while allopurinol did not affect mRNA expression of these uric acid transporters. These findings provide a possible pathophysiological pathway which could explain the higher cardiovascular mortality for febuxostat compared to allopurinol but should be explored further.

Photoannealing of Microtissues Creates High-Density Capillary Network Containing Living Matter in a Volumetric-Independent Manner.

The vascular tree is crucial for the survival and function of large living tissues. Despite breakthroughs in 3D bioprinting to endow engineered tissues with large blood vessels, there is currently no approach to engineer high-density capillary networks into living tissues in a scalable manner. Here, photoannealing of living microtissue (PALM) is presented as a scalable strategy to engineer capillary-rich tissues. Specifically, in-air microfluidics is used to produce living microtissues composed of cell-laden microgels in ultrahigh throughput, which can be photoannealed into a monolithic living matter. Annealed microtissues inherently give rise to an open and interconnected pore network within the resulting living matter. Interestingly, utilizing soft microgels enables microgel deformation, which leads to the uniform formation of capillary-sized pores. Importantly, the ultrahigh throughput nature underlying the microtissue formation uniquely facilitates scalable production of living tissues of clinically relevant sizes (>1 cm3 ) with an integrated high-density capillary network. In short, PALM generates monolithic, microporous, modular tissues that meet the previously unsolved need for large engineered tissues containing high-density vascular networks, which is anticipated to advance the fields of engineered organs, regenerative medicine, and drug screening.

Tracking the dynamics of thrombus formation in a blood vessel-on-chip with visible-light optical coherence tomography.

Thrombus formation is a physiological response to damage in a blood vessel that relies on a complex interplay of platelets, coagulation factors, immune cells, and the vessel wall. The dynamics of thrombus formation are essential for a deeper understanding of many disease processes, like bleeding, wound healing, and thrombosis. However, monitoring thrombus formation is challenging due to the limited imaging options available to analyze flowing blood. In this work, we use a visible-light optical coherence tomography (vis-OCT) system to monitor the dynamic process of the formation of thrombi in a microfluidic blood vessel-on-chip (VoC) device. Inside the VoC, thrombi form in a channel lined with a monolayer of endothelial cells and perfused by human whole blood. We show that the correlation of the vis-OCT signal can be utilized as a marker for thrombus formation. By thresholding the correlation during thrombus formation, we track and quantify the growth of the thrombi over time. We validate our results with fluorescence microscopic imaging of fibrin and platelet markers at the end of the blood perfusion assay. In conclusion, we demonstrate that the correlation of the vis-OCT signal can be used to visualize both the spatial and temporal behavior of the thrombus formation in flowing human whole blood.

Cell Patterning Technology on Polymethyl Methacrylate through Controlled Physicochemical and Biochemical Functionalization.

In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell-material and cell-cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.

Multiplexed fluidic circuit board for controlled perfusion of 3D blood vessels-on-a-chip.

Three-dimensional (3D) blood vessels-on-a-chip (VoC) models integrate the biological complexity of vessel walls with dynamic microenvironmental cues, such as wall shear stress (WSS) and circumferential strain (CS). However, these parameters are difficult to control and are often poorly reproducible due to the high intrinsic diameter variation of individual 3D-VoCs. As a result, the throughput of current 3D systems is one-channel-at-a-time. Here, we developed a fluidic circuit board (FCB) for simultaneous perfusion of up to twelve 3D-VoCs using a single set of control parameters. By designing the internal hydraulic resistances in the FCB appropriately, it was possible to provide a pre-set WSS to all connected 3D-VoCs, despite significant variation in lumen diameters. Using this FCB, we found that variation of CS or WSS induce morphological changes to human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and conclude that control of these parameters using a FCB is necessary to study 3D-VOCs.

Pressure-Driven Perfusion System to Control, Multiplex and Recirculate Cell Culture Medium for Organs-on-Chips.

Organ-on-chip (OoC) devices are increasingly used to mimic the tissue microenvironment of cells in intact organs. This includes microchannels to mimic, for example, fluidic flow through blood vessels. Present methods for controlling microfluidic flow in these systems rely on gravity, rocker systems or external pressure pumps. For many purposes, pressure pumps give the most consistent flow profiles, but they are not well-suited for high throughput as might be required for testing drug responses. Here, we describe a method which allows for multiplexing of microfluidic channels in OoC devices plus the accompanying custom software necessary to run the system. Moreover, we show the approach is also suitable for recirculation of culture medium, an essential cost consideration when expensive culture reagents are used and are not "spent" through uptake by the cells during transient unidirectional flow.

The Effect of Dynamic, In Vivo-like Oxaliplatin on HCT116 Spheroids in a Cancer-on-Chip Model Is Representative of the Response in Xenografts.

The cancer xenograft model in which human cancer cells are implanted in a mouse is one of the most used preclinical models to test the efficacy of novel cancer drugs. However, the model is imperfect; animal models are ethically burdened, and the imperfect efficacy predictions contribute to high clinical attrition of novel drugs. If microfluidic cancer-on-chip models could recapitulate key elements of the xenograft model, then these models could substitute the xenograft model and subsequently surpass the xenograft model by reducing variation, increasing sensitivity and scale, and adding human factors. Here, we exposed HCT116 colorectal cancer spheroids to dynamic, in vivo-like, concentrations of oxaliplatin, including a 5 day drug-free period, on-chip. Growth inhibition on-chip was comparable to existing xenograft studies. Furthermore, immunohistochemistry showed a similar response in proliferation and apoptosis markers. While small volume changes in xenografts are hard to detect, in the chip-system, we could observe a temporary growth delay. Lastly, histopathology and a pharmacodynamic model showed that the cancer spheroid-on-chip was representative of the proliferating outer part of a HCT116 xenograft, thereby capturing the major driver of the drug response of the xenograft. Hence, the cancer-on-chip model recapitulated the response of HCT116 xenografts to oxaliplatin and provided additional drug efficacy information.

Aneurysm-on-a-Chip: Setting Flow Parameters for Microfluidic Endothelial Cultures Based on Computational Fluid Dynamics Modeling of Intracranial Aneurysms.

Intracranial aneurysms are pouch-like extrusions from the vessels at the base of the brain which can rupture and cause a subarachnoid hemorrhage. The pathophysiological mechanism of aneurysm formation is thought to be a consequence of blood flow (hemodynamic) induced changes on the endothelium. In this study, the results of a personalized aneurysm-on-a-chip model using patient-specific flow parameters and patient-specific cells are presented. CT imaging was used to calculate CFD parameters using an immersed boundary method. A microfluidic device either cultured with human umbilical vein endothelial cells (HUVECs) or human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) was used. Both types of endothelial cells were exposed for 24 h to either 0.03 Pa or 1.5 Pa shear stress, corresponding to regions of low shear and high shear in the computational aneurysm model, respectively. As a control, both cell types were also cultured under static conditions for 24 h as a control. Both HUVEC and hiPSC-EC cultures presented as confluent monolayers with no particular cell alignment in static or low shear conditions. Under high shear conditions HUVEC elongated and aligned in the direction of the flow. HiPSC-EC exhibited reduced cell numbers, monolayer gap formation and cells with aberrant, spread-out morphology. Future research should focus on hiPSC-EC stabilization to allow personalized intracranial aneurysm models.

Systematic characterization of cleanroom-free fabricated macrovalves, demonstrating pumps and mixers for automated fluid handling tuned for organ-on-chip applications.

Integrated valves enable automated control in microfluidic systems, as they can be applied for mixing, pumping and compartmentalization purposes. Such automation would be highly valuable for applications in organ-on-chip (OoC) systems. However, OoC systems typically have channel dimensions in the range of hundreds of micrometers, which is an order of magnitude larger than those of typical microfluidic valves. The most-used fabrication process for integrated, normally open polydimethylsiloxane (PDMS) valves requires a reflow photoresist that limits the achievable channel height. In addition, the low stroke volumes of these valves make it challenging to achieve flow rates of microliters per minute, which are typically required in OoC systems. Herein, we present a mechanical 'macrovalve' fabricated by multilayer soft lithography using micromilled direct molds. We demonstrate that these valves can close off rounded channels of up to 700 µm high and 1000 µm wide. Furthermore, we used these macrovalves to create a peristaltic pump with a pumping rate of up to 48 µL/min and a mixing and metering device that can achieve the complete mixing of a volume of 6.4 µL within only 17 s. An initial cell culture experiment demonstrated that a device with integrated macrovalves is biocompatible and allows the cell culture of endothelial cells over multiple days under continuous perfusion and automated medium refreshment.

Fluidic circuit board with modular sensor and valves enables stand-alone, tubeless microfluidic flow control in organs-on-chips.

Organs-on-chips are a unique class of microfluidic in vitro cell culture models, in which the in vivo tissue microenvironment is mimicked. Unfortunately, their widespread use is hampered by their operation complexity and incompatibility with end-user research settings. To address these issues, many commercial and non-commercial platforms have been developed for semi-automated culture of organs-on-chips. However, these organ-on-chip culture platforms each represent a closed ecosystem, with very little opportunity to interchange and integrate components from different platforms or to develop new ones. The translational organ-on-chip platform (TOP) is a multi-institutional effort to develop an open platform for automated organ-on-chip culture and integration of components from various developers. Central to TOP is the fluidic circuit board (FCB), a microfluidic plate with the form factor of a typical well plate. The FCB enables microfluidic control of multiple components like sensors or organ-on-chip devices through an interface based on openly available standards. Here, we report an FCB to integrate commercial and in-house developed components forming a stand-alone flow control system for organs-on-chips. The control system is able to achieve constant and pulsatile flow recirculation through a connected organ-on-chip device. We demonstrate that this system is able to automatically perfuse a heart-on-chip device containing co-cultures of cardiac tissues derived from human pluripotent stem cell-derived cardiomyocytes and monolayers of endothelial cells for five days. Altogether, we conclude that open technology platforms allow the integration of components from different sources to form functional and fit-for-purpose organ-on-chip systems. We anticipate that open platforms will play a central role in catalyzing and maturing further technological development of organ-on-chip culture systems.

Facilitating implementation of organs-on-chips by open platform technology.

Organ-on-chip (OoC) and multi-organs-on-chip (MOoC) systems have the potential to play an important role in drug discovery, disease modeling, and personalized medicine. However, most devices developed in academic labs remain at a proof-of-concept level and do not yet offer the ease-of-use, manufacturability, and throughput that are needed for widespread application. Commercially available OoC are easier to use but often lack the level of complexity of the latest devices in academia. Furthermore, researchers who want to combine different chips into MOoC systems are limited to one supplier, since commercial systems are not compatible with each other. Given these limitations, the implementation of standards in the design and operation of OoCs would strongly facilitate their acceptance by users. Importantly, the implementation of such standards must be carried out by many participants from both industry and academia to ensure a widespread acceptance and adoption. This means that standards must also leave room for proprietary technology development next to promoting interchangeability. An open platform with standardized interfacing and user-friendly operation can fulfill these requirements. In this Perspective article, the concept of an open platform for OoCs is defined from a technical perspective. Moreover, we discuss the importance of involving different stakeholders in the development, manufacturing, and application of such an open platform.

Peristaltic on-chip pump for tunable media circulation and whole blood perfusion in PDMS-free organ-on-chip and Organ-Disc systems.

Organ-on-chip (OoC) systems have become a promising tool for personalized medicine and drug development with advantages over conventional animal models and cell assays. However, the utility of OoCs in industrial settings is still limited, as external pumps and tubing for on-chip fluid transport are dependent on error-prone, manual handling. Here, we present an on-chip pump for OoC and Organ-Disc systems, to perfuse media without external pumps or tubing. Peristaltic pumping is implemented through periodic compression of a flexible pump layer. The disc-shaped, microfluidic module contains four independent systems, each lined with endothelial cells cultured under defined, peristaltic perfusion. Both cell viability and functionality were maintained over several days shown by supernatant analysis and immunostaining. Integrated, on-disc perfusion was further used for cytokine-induced cell activation with physiologic cell responses and for whole blood perfusion assays, both demonstrating the versatility of our system for OoC applications.

Inhibition of Phosphodiesterase 3A by Cilostazol Dampens Proinflammatory Platelet Functions.

OBJECTIVE: platelets possess not only haemostatic but also inflammatory properties, which combined are thought to play a detrimental role in thromboinflammatory diseases such as acute coronary syndromes and stroke. Phosphodiesterase (PDE) 3 and -5 inhibitors have demonstrated efficacy in secondary prevention of arterial thrombosis, partially mediated by their antiplatelet action. Yet it is unclear whether such inhibitors also affect platelets' inflammatory functions. Here, we aimed to examine the effect of the PDE3A inhibitor cilostazol and the PDE5 inhibitor tadalafil on platelet function in various aspects of thromboinflammation. Approach and results: cilostazol, but not tadalafil, delayed ex vivo platelet-dependent fibrin formation under whole blood flow over type I collagen at 1000 s-1. Similar results were obtained with blood from Pde3a deficient mice, indicating that cilostazol effects are mediated via PDE3A. Interestingly, cilostazol specifically reduced the release of phosphatidylserine-positive extracellular vesicles (EVs) from human platelets while not affecting total EV release. Both cilostazol and tadalafil reduced the interaction of human platelets with inflamed endothelium under arterial flow and the release of the chemokines CCL5 and CXCL4 from platelets. Moreover, cilostazol, but not tadalafil, reduced monocyte recruitment and platelet-monocyte interaction in vitro. CONCLUSIONS: this study demonstrated yet unrecognised roles for platelet PDE3A and platelet PDE5 in platelet procoagulant and proinflammatory responses.

Highly parallelized human embryonic stem cell differentiation to cardiac mesoderm in nanoliter chambers on a microfluidic chip.

Human stem cell-derived cells and tissues hold considerable potential for applications in regenerative medicine, disease modeling and drug discovery. The generation, culture and differentiation of stem cells in low-volume, automated and parallelized microfluidic chips hold great promise to accelerate the research in this domain. Here, we show that we can differentiate human embryonic stem cells (hESCs) to early cardiac mesodermal cells in microfluidic chambers that have a volume of only 30 nanoliters, using discontinuous medium perfusion. 64 of these chambers were parallelized on a chip which contained integrated valves to spatiotemporally isolate the chambers and automate cell culture medium exchanges. To confirm cell pluripotency, we tracked hESC proliferation and immunostained the cells for pluripotency markers SOX2 and OCT3/4. During differentiation, we investigated the effect of different medium perfusion frequencies on cell reorganization and the expression of the early cardiac mesoderm reporter MESP1mCherry by live-cell imaging. Our study demonstrates that microfluidic technology can be used to automatically culture, differentiate and study hESC in very low-volume culture chambers even without continuous medium perfusion. This result is an important step towards further automation and parallelization in stem cell technology.

Mimicking and surpassing the xenograft model with cancer-on-chip technology.

Organs-on-chips are in vitro models in which human tissues are cultured in microfluidic compartments with a controlled, dynamic micro-environment. Specific organs-on-chips are being developed to mimic human tumors, but the validation of such 'cancer-on-chip' models for use in drug development is hampered by the complexity and variability of human tumors. An important step towards validation of cancer-on-chip technology could be to first mimic cancer xenograft models, which share multiple characteristics with human cancers but are significantly less complex. Here we review the relevant biological characteristics of a xenograft tumor and show that organ-on-chip technology is capable of mimicking many of these aspects. Actual comparisons between on-chip tumor growth and xenografts are promising but also demonstrate that further development and empirical validation is still needed. Validation of cancer-on-chip models to xenografts would not only represent an important milestone towards acceptance of cancer-on-chip technology, but could also improve drug discovery, personalized cancer medicine, and reduce animal testing.

Organ-on-a-chip technology: a novel approach to investigate cardiovascular diseases.

The development of organs-on-chip (OoC) has revolutionized in vitro cell-culture experiments by allowing a better mimicry of human physiology and pathophysiology that has consequently led researchers to gain more meaningful insights into disease mechanisms. Several models of hearts-on-chips and vessels-on-chips have been demonstrated to recapitulate fundamental aspects of the human cardiovascular system in the recent past. These 2D and 3D systems include synchronized beating cardiomyocytes in hearts-on-chips and vessels-on-chips with layer-based structures and the inclusion of physiological and pathological shear stress conditions. The opportunities to discover novel targets and to perform drug testing with chip-based platforms have substantially enhanced, thanks to the utilization of patient-derived cells and precise control of their microenvironment. These organ models will provide an important asset for future approaches to personalized cardiovascular medicine and improved patient care. However, certain technical and biological challenges remain, making the global utilization of OoCs to tackle unanswered questions in cardiovascular science still rather challenging. This review article aims to introduce and summarize published work on hearts- and vessels-on chips but also to provide an outlook and perspective on how these advanced in vitro systems can be used to tailor disease models with patient-specific characteristics.

Collagen I Based Enzymatically Degradable Membranes for Organ-on-a-Chip Barrier Models.

Organs-on-chips are microphysiological in vitro models of human organs and tissues that rely on culturing cells in a well-controlled microenvironment that has been engineered to include key physical and biochemical parameters. Some systems contain a single perfused microfluidic channel or a patterned hydrogel, whereas more complex devices typically employ two or more microchannels that are separated by a porous membrane, simulating the tissue interface found in many organ subunits. The membranes are typically made of synthetic and biologically inert materials that are then coated with extracellular matrix (ECM) molecules to enhance cell attachment. However, the majority of the material remains foreign and fails to recapitulate the native microenvironment of the barrier tissue. Here, we study microfluidic devices that integrate a vitrified membrane made of collagen-I hydrogel (VC). The biocompatibility of this membrane was confirmed by growing a healthy population of stem cell derived endothelial cells (iPSC-EC) and immortalized retinal pigment epithelium (ARPE-19) on it and assessing morphology by fluorescence microscopy. Moreover, VC membranes were subjected to biochemical degradation using collagenase II. The effects of this biochemical degradation were characterized by the permeability changes to fluorescein. Topographical changes on the VC membrane after enzymatic degradation were also analyzed using scanning electron microscopy. Altogether, we present a dynamically bioresponsive membrane integrated in an organ-on-chip device with which disease-related ECM remodeling can be studied.

Microfluidic organ-on-a-chip model of the outer blood-retinal barrier with clinically relevant read-outs for tissue permeability and vascular structure.

The outer blood-retinal barrier (oBRB) tightly controls the transport processes between the neural tissue of the retina and the underlying blood vessel network. The barrier is formed by the retinal pigment epithelium (RPE), its basal membrane and the underlying choroidal capillary bed. Realistic three-dimensional cell culture based models of the oBRB are needed to study mechanisms and potential treatments of visual disorders such as age-related macular degeneration that result from dysfunction of the barrier tissue. Ideally, such models should also include clinically relevant read-outs to enable translation of experimental findings in the context of pathophysiology. Here, we report a microfluidic organ-on-a-chip model of the oBRB that contains a monolayer of human immortalized RPE and a microvessel of human endothelial cells, separated by a semi-permeable membrane. Confluent monolayers of both cell types were confirmed by fluorescence microscopy. The three-dimensional vascular structures within the chip were imaged by optical coherence tomography: a medical imaging technique, which is routinely applied in ophthalmology. Differences in diameters and vessel density could be readily detected. Upon inducing oxidative stress by treating with hydrogen peroxide (H2O2), a dose dependent increase in barrier permeability was observed by using a dynamic assay for fluorescence tracing, analogous to the clinically used fluorescence angiography. This organ-on-a-chip of the oBRB will allow future studies of complex disease mechanisms and treatments for visual disorders using clinically relevant endpoints in vitro.

Measuring Both pH and O2 with a Single On-Chip Sensor in Cultures of Human Pluripotent Stem Cell-Derived Cardiomyocytes to Track Induced Changes in Cellular Metabolism.

In vitro studies which focus on cellular metabolism can benefit from time-resolved readouts from the living cells. pH and O2 concentration are fundamental parameters upon which cellular metabolism is often inferred. This work demonstrates a novel use of a ruthenium oxide (RuOx) electrode for in vitro studies. The RuOx electrode was characterized to measure both pH and O2 using two different modes. When operated potentiometrically, continuous pH reading can be obtained, and O2 concentration can be measured chronoamperometrically. In this work, we demonstrate the use of the RuOx electrodes in inferring two different types of metabolism of human pluripotent stem cell-derived cardiomyocytes. We also show and discuss the interpretation of the measurements into meaningful extracellular acidification rates and oxygen consumption rates of the cells. Overall, we present the RuOx electrode as a versatile and powerful tool in in vitro cell metabolism studies, especially in comparative settings.