A Microfluidic Blood Vessel-On-Chip Model of Thrombosis

Abstract

BackgroundThrombus formation is regulated by the interplay between endothelial cells (EC), platelets and coagulation factors. However, most in vitro assays used to study thrombosis and develop anti-thrombotic therapies do not include the endothelium. ObjectiveTo develop a thrombus-on-chip model that includes endothelium and whole blood and allows for manipulation of extracellular matrix (ECM), shear stress and the addition of multiple cell types. MethodsA cylindrical vessel was created in a collagen matrix using the needle-based fabrication technique in a microfluidic device. Human umbilical vein EC (HUVEC) or endothelial colony-forming cells (ECFC) were cultured in the channel, with continuous monodirectional media turnover. Immunostaining and permeability measurement validated confluence and integrity of the monolayer. To investigate thrombosis, whole blood from healthy donors was perfused through TNF--activated or untreated EC-lined vessels. Image analysis and time-lapse microscopy were used to quantify labelled platelet adhesion and fibrin deposition. ResultsTNF- treatment resulted in increased platelet adhesion and fibrin deposition compared to control vessels. TNF-induced endothelial activation was confirmed by upregulation of adhesion molecules ICAM-1, E-selectin and tissue factor (TF). Thrombus formation in TNF- treated vessels was inhibited by an anti-TF antibody. In ECFC vessels, platelet adhesion and fibrin deposition were comparable to HUVEC, supporting feasibility of patient-based studies. ConclusionsWe developed a perfused thrombus-on-chip model that combines key elements of thrombus formation including endothelium. The model is amenable to independent control of microenvironmental stimuli, crosstalk with tissue-specific cells, and the inclusion of patients own cells and blood for precision medicine studies. ESSENTIALS- The endothelium is a key contributor to thrombosis - Standard in vitro methods to study thrombosis do not include the endothelium - We have developed a thrombus-on-a-chip model of thromboinflammation to measure live platelet adhesion and fibrin deposition on activated endothelium - Using endothelial colony forming cells (ECFC), we show that the method is suitable for patient studies with autologous endothelium and blood