Developing a 3D blood vessel-on-a-chip microfluidic in vitro model to study thrombosis in a controlled microenvironment

Abstract

Endothelial cells (EC) experience a heterogeneous microenvironment in vivo, which regulates EC phenotype and function. The three main groups of microenvironmental stimuli are: extracellular matrix (ECM), shear stress and crosstalk with tissue-specific cells. In vitro ECs are commonly studied as 2D monolayers on plastic, which cannot mimic this complex microenvironment, limiting the potential for tissue-specific disease modelling. In vivo models can capture all these microenvironmental parameters but cannot be easily manipulated; furthermore, there are pathophysiological differences between animals and humans. Significant progress has been made in the field of microfluidics to develop physiologically relevant in vitro models that can incorporate the endothelial microenvironment to allow disease modelling with human cells. We have developed a 3D blood vessel-on-a-chip microfluidic platform for studying ECs in health and disease, which allows independent control of ECM, shear stress and co-culture with tissue-specific cells. The custom-designed platform comprises a microfluidic device and 3D printed perfusion unit and supports both molecular and functional assays. A cylindrical vessel was created inside the microfluidic device by polymerising collagen around a needle and five chips could be cultured in parallel by attachment to the perfusion unit, which supports real-time imaging. Vessel formation was validated by immunofluorescence and by quantifying permeability. The blood vessel-on-a-chip model was used to study coagulation in real time by perfusing whole blood through the vessel following activation with pro-inflammatory cytokines. Labelled fibrinogen and anti-platelet antibodies were used to visualise thrombus formation. We showed significant increase in platelet adhesion and fibrinogen deposition following endothelial activation, compared to controls, for two different types of ECs. Blocking experiments identified tissue factor and thrombin as determinants of thrombosis in this model. Thus, we have demonstrated that the blood vessel-on-a-chip model provides a novel platform for in vitro studies on the coagulation cascade and mechanisms of thrombosis.