A microengineered vascularized bleeding model that integrates the principal components of hemostasis

Abstract

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an “endothelialized” microfluidic system coupled with a microengineered pneumatic valve that induces a vascular “injury”. With perfusion of whole blood, hemostatic plug formation is visualized and “in vitro bleeding time” is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.

Localized endothelial‐based control of platelet aggregation and coagulation under flow: A proof‐of‐principle vessel‐on‐a‐chip study

“…Our approach herein differs from other vessel‐on‐a‐chip methods, mostly using a confluent monolayer of endothelial cells, which is activated with tumor necrosis factor‐α or is damaged mechanically or by FeCl 3 . Those methods can be considered to reflect conditions of inflammation or severe endothelial injury and may lack the presence of a “healthy” endothelium.…”

“…Cells were then subjected to perfusion with whole blood (DiOC 6 -labeled platelets), and for 10 minutes with recalcified plasma (AF546-fibrinogen), as in Figure 2 activated with tumor necrosis factor-α 13,26 or is damaged mechanically or by FeCl 3 . 15,16 Those methods can be considered to reflect conditions of inflammation or severe endothelial injury and may lack the presence of a "healthy" endothelium. Markedly, we find that the immediate presence of nonstimulated endothelial cells prevents platelet deposition and delays coagulation, in terms of fibrin fiber formation originating from platelet thrombi on noncovered collagen and TF areas.…”

“…Since the 1990s, flow chamber models with confluent endothelial cell monolayers have shown that prolonged stimulation of these cells with tumor necrosis factor‐α results in the exposure of TF (stimulating coagulation) and in the release of multimeric von Willebrand factor (stimulating platelet adhesion) . In recent years, other vascularized microfluidics channels have been developed to also assess the consequences of more severe endothelial disruption, for instance by the free radical‐generating FeCl 3 or by transchannel fluid pressure …”

“…13,14 In recent years, other vascularized microfluidics channels have been developed to also assess the consequences of more severe endothelial disruption, for instance by the free radical-generating FeCl 3 15 or by transchannel fluid pressure. 16 In this paper, we developed a different microfluidic model, using nonconfluent endothelial cells grown on physiologically relevant blood-activating surfaces. We reasoned that the vascular media and atherosclerotic plaques contain high levels of pro-hemostatic collagens as well as TF.…”

Vascularized Microfluidics and the Blood–Endothelium Interface

“…As the primary role of platelets, thrombosis and hemostasis has been the dominant focus of perfusable microfluidic model translation. In an effort to make a clinically relevant model of bleeding to study this process, Sakurai et al [134] developed the first microfluidic bleeding model that recapitulates key aspects of in vivo mechanical injury at the microvascular level. Using a PDMS-based model, a pneumatic valve was introduced that induces vascular injury when engaged.…”