Pulmonary thrombosis is a significant cause of patient mortality; however, there are no effective in vitro models of thrombi formation in human lung microvessels that could also assess therapeutics ...and toxicology of antithrombotic drugs. Here, we show that a microfluidic lung alveolus‐on‐a‐chip lined by human primary alveolar epithelium interfaced with endothelium and cultured under flowing whole blood can be used to perform quantitative analysis of organ‐level contributions to inflammation‐induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet‐endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor‐1 (PAR‐1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development.
Organs-on-chips are microfluidic systems with controlled, dynamic microenvironments in which cultured cells exhibit functions that emulate organ-level physiology. They can in principle be ...'personalised' to reflect individual physiology, for example by including blood samples, primary human tissue, and cells derived from induced pluripotent stem cell-derived cells, as well as by tuning key physico-chemical parameters of the cell culture microenvironment based on personal health data. The personalised nature of such systems, combined with physiologically relevant read-outs, provides new opportunities for person-specific assessment of drug efficacy and safety, as well as personalised strategies for disease prevention and treatment; together, this is known as 'precision medicine'. There are multiple reports of how to personalise organs-on-chips, with examples including airway-on-a-chip systems containing primary patient alveolar epithelial cells, vessels-on-chips with shapes based on personal biomedical imaging data and lung-on-a-chip systems that can be exposed to various regimes of cigarette smoking. In addition, multi-organ chip systems even allow the systematic and dynamic integration of more complex combinations of personalised cell culture parameters. Current personalised organs-on-chips have not yet been used for precision medicine as such. The major challenges that affect the implementation of personalised organs-on-chips in precision medicine are related to obtaining access to personal samples and corresponding health data, as well as to obtaining data on patient outcomes that can confirm the predictive value of personalised organs-on-chips. We argue here that involving all biomedical stakeholders from clinicians and patients to pharmaceutical companies will be integral to transition personalised organs-on-chips to precision medicine.
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.
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.
The vascular endothelium and shear stress are critical determinants of physiological hemostasis and platelet function
in vivo
, yet current diagnostic and monitoring devices do not fully incorporate ...endothelial function under flow in their assessment and, therefore, they can be unreliable and inaccurate. It is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust. Here, we describe a microfluidic device that is lined by a human endothelium that is chemically fixed, but still retains its ability to modulate hemostasis under continuous flow
in vitro
even after few days of storage. This device lined with a fixed endothelium supports formation of platelet-rich thrombi in the presence of physiological shear, similar to a living arterial vessel. We demonstrate the potential clinical value of this device by showing that thrombus formation and platelet function can be measured within minutes using a small volume (0.5 mL) of whole blood taken from subjects receiving antiplatelet medications. The inclusion of a fixed endothelial microvessel will lead to biomimetic analytical devices that can potentially be used for diagnostics and point-of-care applications.
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. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm
2
(± 0.9 Ω.cm
2
) and 28.2 Ω.cm
2
(± 1.3 Ω.cm
2
) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm
2
. Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm
2
. This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.
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.
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.
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