Bioprinted Micro-Clots for Kinetic Analysis of Endothelial Cell-Mediated Fibrinolysis.

Abstract

Vascular hypo-fibrinolysis is a historically underappreciated and understudied aspect of venous thromboembolism (VTE). This paper describes the development of a micro-clot dissolution assay for quantifying the fibrinolytic capacity of endothelial cells - a key driver of VTE development. This assay is enabled using aqueous two-phase systems (ATPS) to bioprint microscale fibrin clots over human umbilical vein endothelial cells (HUVECs). Importantly, these micro-clots are orders of magnitude smaller than conventional fibrin constructs and allow HUVEC-produced plasminogen activators to mediate visually quantifiable fibrinolysis. Using live-cell time-lapse imaging, micro-clot dissolution by HUVECs is tracked, and fibrinolysis kinetics are quantified. The sensitivity of cell-driven fibrinolysis to various stimuli is rapidly tested. The physiological relevance of this convenient high-throughput assay is illustrated through treatments with lipopolysaccharide (LPS) and rosuvastatin that elicit anti- and pro-fibrinolytic responses, respectively. Furthermore, treatment with baricitinib, an anti-inflammatory therapeutic found to increase cardiovascular risks after market approval, provokes an anti-fibrinolytic response - which highlights the potential role of endothelial cells in increasing VTE risk for patients receiving this drug. This endothelial cell fibrinolysis assay provides a high-throughput and versatile drug testing platform - potentially allowing for early preclinical identification of therapeutics that may beneficially enhance or adversely impair endothelial fibrinolysis.