Modelling of thrombus dynamics in a microfluidic 'vasculature'

Why did we fund this project?

This award aims to replace the use of some animal models in haemostasis research by developing a 3D microfluidic “thrombus-on-a-chip” model using whole blood and human cells.

The formation and breakdown of blood clots (haemostasis) is vital for life to prevent blood loss but if these systems become aberrant, they can promote thrombosis and cardiovascular disease. Under normal conditions once wound healing has occurred the body uses its own clot busting system ‘fibrinolysis’ to remove the clot and allow normal blood flow through the vessel. A range of in vivo and in vitro models are used to understand haemostasis and design more effective antithrombotic drugs. Dr Nicola Mutch and Dr Claire Whyte (Co-Supervisor) have previously developed the first thrombus-on-a-chip that allows real-time analysis of fibrinolysis using human blood. However, while this model includes the circulating cells and shear stress of blood flow, it does not include the endothelial cells that line the vasculature in vivo. These cells are crucial in fibrinolysis providing many proteins involved in the pathway and a surface to assemble the various factors.   

The student will further develop the thrombus-on-a-chip model by introducing an endothelial layer using an immortalised cell line. They will then optimise the formation and degradation of a blood clot within the model and monitor the full process using confocal microscopy. The student will develop skills in cell culture, transfection and biochemical and activity assays.

This Studentship was co-awarded with the British Heart Foundation (BHF).

Haemostasis, the arrest of blood flow, is essential for life, with deficiencies in essential proteins or cells causing life threatening bleeding conditions in afflicted individuals. However, if these systems become aberrant, they result in deleterious thrombotic complications in the arterial and venous circulation accounting for more than one third of deaths in the Western world. Blood clot formation is countered by a process called, fibrinolysis, which is nature’s way of restricting the size of a clot thereby preventing occlusion of blood vessels. Our knowledge on fibrinolysis is impacted by ineffective systems and methodology that do not accurately reflect the process in vivo. Yet, understanding these mechanisms is crucial in our fight against thromboembolic disease and would permit the design of more effective drugs that promote breakdown of blood clots in pathogenic situations, such as during stroke, deep vein thrombosis and coronary heart disease, while limiting the bleeding complications associated with current antithrombotic drugs.

Animal models have been the mainstay in the study of haemostasis and thrombosis as these systems are complex and are impacted by many cell types and the shear stress of the vasculature, which alters the structure and composition of the clot. In addition, genetic mouse models are abundant, and the mouse is considered a good model to study blood clot formation as it is similar to the human system. Within the UK institutes conducting animal research into thrombosis and haemostasis would use in excess of 2000 mice per annum. In recent years several ex vivo models of platelet activation and coagulation have been developed and have now firmly established themselves within the thrombosis and haemostasis community. Combined with advances in molecular biology techniques to manipulate expression of key proteins this has led to replacement and reduction of animal usage in this field. However, these models do not include steps to visualise thrombus stability and degradation. Our laboratory was the first to develop an ex vivo model with human whole blood in which to study fibrinolysis. This model recapitulates the shear stress of the vasculature and incorporates circulating cells within the blood that impact on thrombus formation and stability, including platelets, monocytes and neutrophils. A major limitation of this current model is that it lacks the endothelial surface that lines the vasculature in vivo and supplies many factors that influence fibrinolysis in vivo.

The fundamental objective of this study is to develop a thrombus-on-a-chip model that encompasses all elements of the vasculature, including the endothelial layer. This model will be the first to permit real-time analysis of fibrinolytic activity within the thrombus environment in a microcirculation akin to the vasculature. Use of this 3D microfluidic model will replace the use of animals to study fibrinolysis within our own institution, cutting the number of mice by 1800 per year. Importantly, if adopted by other institutions this model would significantly reduce the number of animals used for thrombosis and haemostasis research by up to 1000-2000 per institution. We will ensure that the methodology is published within scientific journals and on the NC3Rs gateway to encourage uptake and will present the model and National and International conferences. We envisage that this sophisticated thrombus-on-a-chip model will enhance our understanding of the fibrinolytic process and be used as a platform to test novel antithrombotic drugs.