A Microfluidic Blood Vessel-On-Chip Model of Thrombosis

Background

Thrombus 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.

Objective

To 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.

Methods

A 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-α-activated or untreated EC-lined vessels. Image analysis and time-lapse microscopy were used to quantify labelled platelet adhesion and fibrin deposition.

Results

TNF-α 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-α 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.

Conclusions

We 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