Bioprinted Micro‐Clots for Kinetic Analysis of Endothelial Cell‐Mediated Fibrinolysis

This paper describes the development of a microscale model that quantifies the breakdown of fibrin by endothelial cells. The small size scale of this engineered system enables fibrin breakdown to be observed without the addition of external activators. This system could help in identifying compounds which both promote and hinder thrombosis.

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.