Arterial thrombosis is the main instigating factor of heart attacks and strokes, which result in over 14 million deaths worldwide every year. The mechanism of thrombosis involves factors from the blood and the vessel wall, and it also relies strongly on 3D vessel geometry and local blood flow patterns. Microfluidic chip-based vascular models allow controlled in vitro studies of the interaction between vessel wall and blood in thrombosis, but until now, they could not fully recapitulate the 3D geometry and blood flow patterns of real-life healthy or diseased arteries. Here we present a method for fabricating microfluidic chips containing miniaturized vascular structures that closely mimic architectures found in both healthy and stenotic blood vessels. By applying stereolithography (SLA) 3D printing of computed tomography angiography (CTA) data, 3D vessel constructs were produced with diameters of 400 μm, and resolution as low as 25 μm. The 3D-printed templates in turn were used as moulds for polydimethylsiloxane (PDMS)-based soft lithography to create microfluidic chips containing miniaturized replicates of in vivo vessel geometries. By applying computational fluid dynamics (CFD) modeling a correlation in terms of flow fields and local wall shear rate was found between the original and miniaturized artery. The walls of the microfluidic chips were coated with human umbilical vein endothelial cells (HUVECs) which formed a confluent monolayer as confirmed by confocal fluorescence microscopy. The endothelialised microfluidic devices, with healthy and stenotic geometries, were perfused with human whole blood with fluorescently labeled platelets at physiologically relevant shear rates. After 15 minutes of perfusion the healthy geometries showed no sign of thrombosis, while the stenotic geometries did induce thrombosis at and downstream of the stenotic area. Overall, the novel methodology reported here, overcomes important design limitations found in typical 2D wafer-based soft lithography microfabrication techniques and shows great potential for controlled studies of the role of 3D vessel geometries and blood flow patterns in arterial thrombosis.

Macrophages are innate immune cells that prevent infections and help in wound healing and vascular inflammation. While these cells are natural helper cells, they also contribute to chronic diseases, e.g., by infiltrating the endothelial layer in early atherosclerosis and by promoting vascular inflammation. There is a crosstalk between inflammatory pathways and key players in thrombosis, such as platelets and endothelial cells – a phenomenon known as ‘thromboinflammation’. The role of the embedded macrophages in thromboinflammation in the context of vascular disease is incompletely understood. Blood vessels-on-chips, which are microfluidic vascular cell culture models, have been used extensively to study aspects of vascular disease, like permeability, immune cell adhesion and thrombosis. Blood perfusion assays in blood vessel-on-chip models benefit from multiple unique aspects of the models, such as control of microvessel structure and well-defined flow patterns, as well as the ability to perform live imaging. However, due to their simplified nature, blood vessels-on-chip models have not yet been used to capture the complex cellular crosstalk that is important in thromboinflammation. Using induced pluripotent stem cell-derived endothelial cells and polarized THP-1 monocytes, we have developed and systematically set up a 3D blood vessel-on-chip with embedded (lipid-laden) macrophages, which is created using sequential cell seeding in viscous finger patterned collagen hydrogels. We have set up a human whole blood perfusion assay for these 3D blood vessels-on-chip. An increased deposition of fibrin in the blood vessel-on-chip models containing lipid-laden macrophages was observed. We anticipate the future use of this advanced vascular in vitro model in drug development for early atherosclerosis or aspects of other vascular diseases. Graphical Abstract

Organ-on-a-chip models with incorporated vasculature are becoming more popular to study platelet biology. A large variety of image analysis techniques are currently used to determine platelet coverage, ranging from manually setting thresholds to scoring platelet aggregates. In this communication, an automated methodology is introduced, which corrects misalignment of a microfluidic channel, automatically defines regions of interest and utilizes a triangle threshold to determine platelet coverages and platelet aggregate size distributions. A comparison between the automated methodology and manual identification of platelet aggregates shows a high accuracy of the triangle methodology. Furthermore, the image analysis methodology can determine platelet coverages and platelet size distributions in microfluidic channels lined with either untreated or activated endothelium used for whole blood perfusion, proving the robustness of the method.

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.