Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium

Abstract

Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils’ importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.

Platelet Mechanobiology Inspired Microdevices: From Hematological Function Tests to Disease and Drug Screening

“… 4) Microfluidics present better biomimetic performance. Endothelialized microfluidics are rapidly evolving as humanized screening platforms ( Albers et al, 2019 ; Jenny et al, 2020 ; Dupuy et al, 2021 ). Increasing evidences demonstrate their great potential in simulating the interplays between platelets and circulatory systems ( Zhang et al, 2021 ).…”

Emerging Microfluidic Approaches for Platelet Mechanobiology and Interplay With Circulatory Systems

“…In the past decade, soft lithography has rapidly grown to enable high-fidelity micropatterning. Parallel straight channels can simply be fabricated by the single-layered photolithography and poly(dimethylsiloxane) (PDMS) casting processes, resulting in a vascular geometry with laminar flow ( 5 , 16 , 41 , 42 ). When connected with external syringe pumps, such simple microfluidic setting enables systematic investigation of shear dependent platelet adhesion ( 43 ) and aggregation ( 44 ).…”

“…When connected with external syringe pumps, such simple microfluidic setting enables systematic investigation of shear dependent platelet adhesion ( 43 ) and aggregation ( 44 ). Additionally, vascular cell cultures can be incorporated; specifically, endothelialized microchannels can model the interaction between blood components and the vessel wall under physiological hemodynamic parameters ( 45 ), or even when subjected to proinflammatory or other endothelial diseased conditions ( 16 ).…”

“…The FeCl 3 influx induced endothelial injury, followed by the binding between Fe 3+ ions and negatively charged proteins, which lead to platelet activation and occlusive aggregation. Dupuy et al developed a thromboinflammation model by using six parallel microchannels with fixed human umbilical vein endothelial cells (HUVECs) alignment ( 16 ). Tumor necrosis factor alpha (TNF-α) treatment stimulated the expression of ICAM-1 and VCAM-1, and the secretion of VWF and tissue factor on the endothelium, leading to a dose-dependent increase in platelet–endothelial binding.…”

“…Increasingly, microfluidic technologies have emerged as powerful and indispensable approaches to investigate the mechanosensitive behaviors of platelets ( 2 , 12 , 14 ). Numerous endothelialized microfluidics have been invented to model the interplay between platelets, endothelium and VWF under physiologically relevant hemodynamic microenvironments ( 6 , 15 , 16 ). In the context of platelet mechanobiology, we summarize these state-of-the-art microfluidic methodologies that have recently been invented in the field.…”