Bioprinted Micro-Clots for Kinetic Analysis of Endothelial Cell-Mediated Fibrinolysis
Breakthrough Research on Micro-Scale Exploration of Thrombolysis Kinetics
Research Background and Problem
Venous thromboembolism (VTE), a severe health condition, causes approximately 500,000 deaths annually in the United States alone. The occurrence of VTE is closely related to the formation and resistance to dissolution of thrombi in veins, commonly termed hypo-fibrinolysis. However, for a long time, VTE research has primarily focused on “hypercoagulability,” the mechanism of thrombus formation, while relatively less attention has been given to hypo-fibrinolysis. Current VTE treatments mainly rely on anticoagulants, which inhibit clot formation and propagation but do not effectively enhance clot dissolution. The only widely adopted fibrinolysis-related treatment, exogenous plasminogen activators (thrombolytics), is limited in use due to the high risk of severe bleeding.
Furthermore, the slow progress in the development of drugs related to hypo-fibrinolysis is partly due to the lack of efficient in vitro experimental models. Most current fibrinolysis experiments use simplified in vitro detection methods, such as monitoring fibrinolysis through turbidity changes after adding plasminogen activators to patient blood samples. However, these methods are too simplified to realistically reflect the multifactorial influences on hypo-fibrinolysis. Additionally, these methods rarely consider the involvement of endothelial cells (ECs), which are crucial regulators of fibrinolysis.
To overcome the limitations of existing methods and knowledge gaps, a research team from Georgia Institute of Technology, Emory University, and Vanderbilt University Medical Center developed a micro-scale thrombosis dissolution kinetic analysis platform based on bioprinting technology. This platform enables a comprehensive analysis of the thrombolytic processes regulated by endothelial cells, further assessing the effects of environmental stimuli and drugs on fibrinolysis. This research was published in the 2025 issue of Advanced Healthcare Materials, with Shuichi Takayama as the corresponding author.
Research Process
1. Construction of the Micro-Thrombolysis Analysis Platform
The research team used aqueous two-phase systems (ATPS) and bioprinting technology to construct micro-scale fibrin clots directly on cultured human umbilical vein endothelial cells (HUVECs). Specifically, thrombin- and fibrinogen-containing two-phase liquids were controlled through ATPS printing to achieve separation, eventually forming uniform and stable micro-scale fibrin clots on the surface of HUVECs. This process faithfully mimics fibrin deposition and coagulation on endothelial cells following vascular injury.
The printed clots are maintained within the micro volumes (microliter or sub-microliter scale) of a 96-well plate, enabling real-time monitoring of the dissolution process. Using the Incucyte automated live-cell imaging technology, researchers continuously monitored the clot dissolution process for 24 hours and quantified the image data through pixel intensity algorithms in MATLAB.
An innovative point of this method is that it does not require exogenous plasminogen activators. It relies entirely on fibrinolytic factors secreted by endothelial cells, such as tissue-type plasminogen activator (TPA), to complete clot degradation, thus realistically reflecting the fibrinolytic capacity under cellular regulation.
2. Dynamic Characteristics of Micro-Thrombolysis
The study shows a nonlinear increase in clot dissolution time with the increase in fibrin clot volume. This is attributed to the relative reduction in the exposed surface area per unit volume in larger clots, limiting interaction with fibrinolytic factors produced by endothelial cells. Furthermore, a lactate dehydrogenase release assay (LDH-Glo Assay) confirmed that cytotoxicity levels remained low throughout the clot dissolution process, demonstrating the physiological compatibility of the assay.
3. Effects of Environmental Stimuli on Fibrinolysis
The research team further tested the effects of different stimuli on endothelial cell-regulated fibrinolysis. Bacterial lipopolysaccharide (LPS) is known to increase the expression of fibrinolysis inhibitors, thereby reducing fibrinolytic capacity. Experiments showed that as LPS concentration increased, clot dissolution time significantly prolonged, and residual fibrin left undissolved increased accordingly. This phenomenon fully reveals the LPS-induced hypo-fibrinolytic state and demonstrates the potential of the experimental platform as a disease model.
4. Drug Testing and Effect Mechanisms
The researchers used this platform to evaluate several drugs with known anticoagulant or fibrinolytic effects: - Rosuvastatin: Statins are known to have pro-fibrinolytic effects, but their mechanisms remain unclear. In experiments involving endothelial cells, rosuvastatin was found to significantly accelerate clot dissolution in a dose-dependent manner. However, this effect disappeared in the absence of endothelial cells, indicating that rosuvastatin promotes fibrinolysis through endothelial cell regulation rather than directly facilitating clot dissolution.
- Baricitinib: As a JAK kinase inhibitor flagged by the FDA for its risk of thrombosis, baricitinib at high concentrations significantly delayed the fibrinolytic process and left small amounts of fibrin undissolved. However, at low concentrations, its effects were weaker and might even display a pro-fibrinolytic effect under inflammatory conditions.
Based on these results, this method can be used for the early screening of drugs that may induce hypo-fibrinolysis risks.
5. Advantages of the Experimental System and Future Directions
The research team also discussed the experimental constraints and future directions. The current micro-thrombus model is primarily based on fibrin networks. In the future, incorporating red blood cells, platelets, and other components can build more complex thrombus environments. Additionally, introducing fluid dynamic models that emulate complex flow states in injured vessels could more precisely simulate fibrinolytic balance under physiological or pathological conditions.
Significance and Value of the Research
Through the innovative micro-thrombolysis analysis platform, this study dynamically recreated the thrombolytic process regulated by endothelial cells. Due to its unique feature of not requiring the addition of exogenous fibrinolytic factors, the platform can authentically reflect changes in fibrinolytic capacity in physiological states. The high-throughput nature and flexible design of the platform also make it widely applicable to drug screening and risk prediction. By evaluating exogenous stimuli such as LPS and baricitinib that induce hypo-fibrinolysis, the platform provides a more detailed analytical tool for studying the pathological mechanisms of hypo-fibrinolysis.
This research not only fills a technological gap in VTE-related hypo-fibrinolysis studies but also opens new prospects for disease treatment and drug development. The development of novel fibrinolysis-modulating drugs is expected to achieve earlier validation and screening through this platform.