Abstract 529: Assessing Hemostasis and Platelet Function Using a Microfluidic Device Lined with Fixed Human Endothelium

Abstract

Mutual signaling between endothelium and platelets is widely recognized as critical for regulation of hemostasis and thrombotic disorders associated with various diseases, such as, arteriosclerosis, sepsis, and diabetes. Yet no reliable and practical assays exist that can measure the effects of cross-talk between platelets and inflamed vessel walls in the presence of physiological shear. Flow chambers have been applied previously to study basic science, but they have not been used in practical settings due to the difficulty in maintaining living cells in devices. Specifically, because it is extremely difficult to maintain living cell cultures for extended times, it would be impossible to maintain the robustness of these assays. Here, we describe a microfluidic device lined by a chemically fixed human endothelium that retains its ability to support thrombus formation in the presence of blood flow for multiple days under standard storage conditions. We coat the inner surface of a rectangular device with collagen and then culture with endothelial cells on all four walls in order to create a vascular tube. Under activation by increasing doses of TNF-α followed by fixation, the device continues to exhibit multiple proinflammatory and adhesion molecules. Correspondingly, a dose-dependent increase in platelet adhesion to the endothelial layer is also observed. Importantly, we do not observe any significant difference in platelet adhesion between living and fixed endothelium. Furthermore, the morphology of thrombi appears similar to that of thrombi that form on living endothelium of a laser-injured mouse in vivo . To establish clinical utility, we also perfused whole blood of patients taking antiplatelet drugs, and found that these subjects showed a significant reduction in platelet adhesion in our devices. In contrast, conventional aggregometry analysis was very time consuming and tedious. Also, on a collagen-coated flow chamber we found no significant difference in platelet coverage between normal controls and patients. Taken together, this study confirms that this microdevice-incorporating fixed endothelium, whole blood and shear stress-can be used as a global hemostasis assay in research as well as many practical and clinical settings.