Andries Dirk van der Meer

Organs-on-chips: breaking the in vitro impasse

In vitro models of biological tissues are indispensable tools for unraveling human physiology and pathogenesis. They usually consist of a single layer of a single cell type, which makes them robust and suitable for parallelized research. However, due to their simplicity, in vitro models are also less valid as true reflections of the complex biological tissues of the human body. Even though the realism of the models can be increased by including more cell types, this will inevitably lead to a decrease in robustness and throughput. The constant trade-off between realism and simplicity has led to an impasse in the development of new in vitro models. Organs-on-chips, a class of microengineered in vitro tissue models, have the potential to break the in vitro impasse. These models combine an artificially engineered, physiologically realistic cell culture microenvironment with the potential for parallelization and increased throughput. They are robust, because the engineered physiological, organ-level features such as tissue organization, geometry, soluble gradients and mechanical stimulation are well-defined and controlled. Moreover, their microfluidic properties and integrated sensors pave the way for high-throughput studies. In this review, we define the in vitro impasse, we explain why organs-on-chips have the potential to break the impasse and we formulate a view on the future of the field. We focus on the design philosophy of organs-on-chips, the integration of technology and biology and on how to connect to the potential end-users.

Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner

Rupture of a vulnerable atherosclerotic plaque causes thrombus formation and precipitates cardiovascular diseases. In addition to the thrombogenic content of a plaque, also the hemodynamic microenvironment plays a major role in thrombus formation. How the altered hemodynamics around a plaque promote pathological thrombus formation is not well understood. In this study, we provide evidence that plaque geometries result in fluid mechanical conditions that promote platelet aggregation and thrombus formation by increased accumulation and activity of von Willebrand factor (vWF) at poststenotic sites. Resonant-scanning multiphoton microscopy revealed that in vivo arterial stenosis of a damaged carotid artery markedly increased platelet aggregate formation in the stenotic outlet region. Complementary in vitro studies using microfluidic stenotic chambers, designed to mimic the flow conditions in a stenotic artery, showed enhanced platelet aggregation in the stenotic outlet region at 60–80% channel occlusion over a range of input wall shear rates. The poststenotic thrombus formation was critically dependent on bloodborne vWF and autocrine platelet stimulation. In stenotic chambers containing endothelial cells, flow provoked increased endothelial vWF secretion in the stenotic outlet region, contributing to exacerbated platelet aggregation. Taken together, this study identifies a role for the shear-sensitive protein vWF in transducing hemodynamic forces that are present around a stenosis to a prothrombogenic microenvironment resulting in spatially confined and exacerbated platelet aggregation in the stenosis outlet region. The developed stenotic microfluidic chamber offers a realistic platform for in vitro evaluation of shear-dependent thrombus formation in the setting of atherosclerosis.

A microfluidic wound-healing assay for quantifying endothelial cell migration

Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-mum-wide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF(165)) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF(165) could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress.

Distinct Contributions of Astrocytes and Pericytes to Neuroinflammation Identified in a 3D Human Blood-Brain Barrier on a Chip

Neurovascular inflammation is a major contributor to many neurological disorders, but modeling these processes in vitro has proven to be difficult. Here, we microengineered a three-dimensional (3D) model of the human blood-brain barrier (BBB) within a microfluidic chip by creating a cylindrical collagen gel containing a central hollow lumen inside a microchannel, culturing primary human brain microvascular endothelial cells on the gel’s inner surface, and flowing medium through the lumen. Studies were carried out with the engineered microvessel containing endothelium in the presence or absence of either primary human brain pericytes beneath the endothelium or primary human brain astrocytes within the surrounding collagen gel to explore the ability of this simplified model to identify distinct contributions of these supporting cells to the neuroinflammatory response. This human 3D BBB-on-a-chip exhibited barrier permeability similar to that observed in other in vitro BBB models created with non-human cells, and when stimulated with the inflammatory trigger, tumor necrosis factor-alpha (TNF-α), different secretion profiles for granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed depending on the presence of astrocytes or pericytes. Importantly, the levels of these responses detected in the 3D BBB chip were significantly greater than when the same cells were co-cultured in static Transwell plates. Thus, as G-CSF and IL-6 have been reported to play important roles in neuroprotection and neuroactivation in vivo, this 3D BBB chip potentially offers a new method to study human neurovascular function and inflammation in vitro, and to identify physiological contributions of individual cell types.

Microfluidic organ-on-chip technology for blood-brain barrier research

ABSTRACT Organs-on-chips are a new class of microengineered laboratory models that combine several of the advantages of current in vivo and in vitro models. In this review, we summarize the advances that have been made in the development of organ-on-chip models of the blood-brain barrier (BBBs-on-chips) and the challenges that are still ahead. The BBB is formed by specialized endothelial cells and separates blood from brain tissue. It protects the brain from harmful compounds from the blood and provides homeostasis for optimal neuronal function. Studying BBB function and dysfunction is important for drug development and biomedical research. Microfluidic BBBs-on-chips enable real-time study of (human) cells in an engineered physiological microenvironment, for example incorporating small geometries and fluid flow as well as sensors. Examples of BBBs-on-chips in literature already show the potential of more realistic microenvironments and the study of organ-level functions. A key challenge in the field of BBB-on-chip development is the current lack of standardized quantification of parameters such as barrier permeability and shear stress. This limits the potential for direct comparison of the performance of different BBB-on-chip models to each other and existing models. We give recommendations for further standardization in model characterization and conclude that the rapidly emerging field of BBB-on-chip models holds great promise for further studies in BBB biology and drug development.

Direct quantification of transendothelial electrical resistance in organs-on-chips

Measuring transendothelial or transepithelial electrical resistance (TEER) is a widely used method to monitor cellular barrier tightness in organs-on-chips. Unfortunately, integrated electrodes close to the cellular barrier hamper visual inspection of the cells or require specialized cleanroom processes to fabricate see-through electrodes. Out-of-view electrodes inserted into the chips outlets are influenced by the fluid-filled microchannels with relatively high resistance. In this case, small changes in temperature or medium composition strongly affect the apparent TEER. To solve this, we propose a simple and universally applicable method to directly determine the TEER in microfluidic organs-on-chips without the need for integrated electrodes close to the cellular barrier. Using four electrodes inserted into two channels - two on each side of the porous membrane - and six different measurement configurations we can directly derive the isolated TEER independent of channel properties. We show that this method removes large variation of non-biological origin in chips filled with culture medium. Furthermore, we demonstrate the use of our method by quantifying the TEER of a monolayer of human hCMEC/D3 cerebral endothelial cells, mimicking the blood-brain barrier inside our microfluidic organ-on-chip device. We found stable TEER values of 22 Ω cm(2)±1.3 Ω cm(2) (average ± standard error of the mean of 4 chips), comparable to other TEER values reported for hCMEC/D3 cells in well-established Transwell systems. In conclusion, we demonstrate a simple and robust way to directly determine TEER that is applicable to any organ-on-chip device with two channels separated by a membrane. This enables stable and easily applicable TEER measurements without the need for specialized cleanroom processes and with visibility on the measured cell layer.

Organ‐on‐Chip Recapitulates Thrombosis Induced by an anti‐CD154 Monoclonal Antibody: Translational Potential of Advanced Microengineered Systems

Clinical development of Hu5c8, a monoclonal antibody against CD40L intended for treatment of autoimmune disorders, was terminated due to unexpected thrombotic complications. These life‐threatening side effects were not discovered during preclinical testing due to the lack of predictive models. In the present study, we describe the development of a microengineered system lined by human endothelium perfused with human whole blood, a “Vessel‐Chip.” The Vessel‐Chip allowed us to evaluate key parameters in thrombosis, such as endothelial activation, platelet adhesion, platelet aggregation, fibrin clot formation, and thrombin anti‐thrombin complexes in the Chip‐effluent in response to Hu5c8 in the presence of soluble CD40L. Importantly, the observed prothrombotic effects were not observed with Hu5c8‐IgG2σ designed with an Fc domain that does not bind the FcγRIIa receptor, suggesting that this approach may have a low potential risk for thrombosis. Our results demonstrate the translational potential of Organs‐on‐Chips, as advanced microengineered systems to better predict human response.

Blood-brain barrier (BBB): an overview of the research of the blood-brain barrier using microfluidic devices

The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. This chapter highlights the anatomy and physiology of the BBB and gives an overview of the current available in vitro models to study the BBB in detail. Proof-of-concept work of BBB-on-Chips are described. Additionally, examples are given to optimize the present devices by engineering the microenvironment to better mimic the in vivo situation. This combination of biomedical science and micro-engineering will generate exciting new results in the field of neurovascular biology.

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

Barriers-on-chips: Measurement of barrier function of tissues in organs-on-chips

Disruption of tissue barriers formed by cells is an integral part of the pathophysiology of many diseases. Therefore, a thorough understanding of tissue barrier function is essential when studying the causes and mechanisms of disease as well as when developing novel treatments. In vitro methods play an integral role in understanding tissue barrier function, and several techniques have been developed in order to evaluate barrier integrity of cultured cell layers, from microscopy imaging of cell-cell adhesion proteins to measuring ionic currents, to flux of water or transport of molecules across cellular barriers. Unfortunately, many of the current in vitro methods suffer from not fully recapitulating the microenvironment of tissues and organs. Recently, organ-on-chip devices have emerged to overcome this challenge. Organs-on-chips are microfluidic cell culture devices with continuously perfused microchannels inhabited by living cells. Freedom of changing the design of device architecture offers the opportunity of recapitulating the in vivo physiological environment while measuring barrier function. Assessment of barriers in organs-on-chips can be challenging as they may require dedicated setups and have smaller volumes that are more sensitive to environmental conditions. But they do provide the option of continuous, non-invasive sensing of barrier quality, which enables better investigation of important aspects of pathophysiology, biological processes, and development of therapies that target barrier tissues. Here, we discuss several techniques to assess barrier function of tissues in organs-on-chips, highlighting advantages and technical challenges.