Methods, Systems, And Compositions For Determining Blood Clot Formation, And Uses Thereof

Abstract

A method is directed to determining a thrombosis function and includes flowing a fluid sample over a surface having a fixed endothelial cell monolayer. The method further includes stimulating the fixed endothelial cell monolayer to induce formation of a clot, the clot being formed via interaction between the fixed endothelial cell monolayer and the fluid sample. In response to the clot formation, the method further includes determining a thrombosis function associated with the fluid sample and the fixed endothelial cell monolayer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to and benefit of U.S. Provisional Patent Application Ser. No. 62 / 165,272, filed on May 22, 2015, and U.S. Provisional Patent Application Ser. No. 62 / 310,166, filed on Mar. 18, 2016, each of which is hereby incorporated by reference herein in its entirety.GOVERNMENT SUPPORT[0002]The invention was made with Government Support under N66001-11-1-4180 awarded by the Space and Naval Warfare Systems Center of the U.S. Department of Defense, and under HR0011-13-C-0025 awarded by the Defense Advanced Research Projects Agency of the U.S. Department of Defense. The government has certain rights in the invention.FIELD OF THE INVENTION[0003]The present invention relates generally to quantifying a thrombosis-related function in vitro based on physiologically relevant conditions, and, more particularly, to a microfluidic system having fluid flow interaction between a fixed endothelial layer and cells (such as platelets)...

AI Technical Summary

Benefits of technology

[0014]According to one aspect of the present invention, a microfluidic device is lined with a human endothelium that is chemically fixed, but still retains its ability to modulate hemostasis under continuous flow in vitro. For example, according to one method, microfluidic channels are seeded with collagen and endothelial cells and are left either untreated or treated with tumor necrosis factor-α (TNF-α). The cells are, then, fixed with formaldehyde. Recalcified citrated whole blood (0.5 mL) from healthy volunteers or patients taking antiplatelet medication is perfused and platelet coverage is recorded. The chemopreserved endothelialized device is lined with a bioinspired material that supports formation of platelet-rich thrombi in the presence of physiological shear, similar to a living arterial vessel. Furthermore, the method demonstrates the potential clinical value of the chemopreserved endothelialized device by showing that thrombus formation and platelet function are measurable within minutes using a small volume of whole blood taken from subjects receiving antiplatelet medications. The method further demonstrates potentially greater reliability than standard platelet function tests and collagen-coated perfusion chambers.

[0015]According to another aspect of the present invention, a microengineered lung-on-chip device is used for studying human pulmonary blood clotting and platelet-endothelial interaction dynamics. The lung-on-chip is a microfluidic device populated with primary alveolar cells (“AE”) localized within a top channel and vascular endothelial cells in a bottom compartment. The top channel and the bottom compartment are separated by a matrix-coated membrane. Whole blood is perfused in the vascular compartment while the epithelium is stimulated with a cytokine or endotoxin, and platelet-endothelial interactions are recorded in real-time. To quantify the dynamics of the platelet-endothelial interactions, a stochastic analytical method is provided that is highly sensitive to changes in endothelial and platelet activation. In vitro, the presence of alveolar epithelium is shown to be beneficial for reconstituting pulmonary thrombosis in response to an inflammatory stimulus of lipopolysaccharide (“LPS”). Additionally, this model is used in drug development by analyzing the effect of a novel protease activator receptor-1 (“PAR1”) antithrombotic compound, termed parmodulin 2 (“PM2”), and demonstrate that PM2 has an endothelial cytoprotective effect in response to LPS-mediated inflammation. The lung-on-chip device reconstitutes organ-level functionality that accurately reflects many aspects of human pulmonary thrombosis and appears to offer a valuable platform for drug development.

[0046]The methods and / or systems described herein can provide tools to diagnose a disease or disorder induced by cell dysfunction or abnormal cell-cell interaction in a subject. Accordingly, another aspect described herein relates to a method of determining if a subject is at risk, or has, a disease or disorder induced by cell dysfunction or abnormal cell-cell interaction. The method comprises: (a) flowing a fluid sample of the subject over a surface comprising a fixed cell monolayer thereon; (b) detecting interaction of cells in the fluid sample between each other and / or with the fixed cell monolayer; and (d) identifying the subject to be at risk, or have the disease or disorder induced by cell dysfunction when the cell-cell interaction is higher than a control; or identifying the subject to be less likely to have a disease or disorder induced by cell dysfunction when the cell-cell interaction is no more than the control.

Problems solved by technology

Generally, the vascular endothelium and shear stress are critical determinants of hemostasis and platelet function in vivo, and yet, current diagnostic and monitoring devices do not fully incorporate endothelial function under flow in their assessment.

Therefore, current diagnostic and monitoring devices can be unreliable and inaccurate.

Furthermore, it is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust.

Yet, no practical diagnostic assays exist that can measure cross-talk between platelets and inflamed vessel walls in the presence of physiological shear.

While these devices have been very useful in advancing research, they have not been used in clinical settings due to the difficulty in maintaining living endothelial cells in them.

Specifically, because it is extremely difficult to maintain the viability of living cell cultures for extended times in non-controlled settings, it is virtually impossible to rely on these assays.

Therefore, the only microfluidic devices that are currently being deployed in clinical diagnostic settings are lined with collagen to mimic thrombus formation and platelet aggregation induced in response to vascular injury, and, thus, they fail to capture the physiological interplay between endothelial cells, platelets and fluid shear stress that is so relevant to hemostasis in inflammatory diseases.

Additionally, pulmonary microvascular thrombosis is a catastrophic condition amounting to a large number of patient deaths worldwide.

Despite significant progress in understanding fundamental biology of lung hemostasis and thrombosis, it is still very difficult to predict response and study mechanism of action of potential drug candidates to humans.

This is partly so because currently available in vitro assays do not recapitulate physiologically-relevant forces, such as shear stress, and animal models can be very complex allowing limited experimental manipulation, making it impossible to dissect and study intercellular signaling.

Although epithelial injury, endothelial dysfunction, and in situ thrombotic lesions are observed often in human patients in chronic pulmonary diseases, animal models of pulmonary dysfunction are still unable to completely mimic the altered hemostasis and hemodynamic complexity of the lung.

Importantly, animal models can be very complex and it may be impossible to study cell-cell interactions between multiple tissues independently of each other during blood clotting or drug administration.

In vitro, commercially available coagulation and platelet function technologies also have serious limitations due to the fact that they do not incorporate physiological tissue-tissue or cell-cell interactions, and relevant fluid dynamics of blood cells, which are key determinants of thrombosis.

In research laboratories, dishes and transwell plates have been used for decades to culture cells and study basic biology, but these are static systems, highly non-physiological and cannot recapitulate tissue or organ-level functionality.

However, being macroscale devices, these chambers do not mimic small blood vessels, typically do not incorporate endothelium, and require large blood sample volumes for analysis.

However, these devices are also limited in studying organ-level pulmonary thrombosis, in part because they do not include the role of live epithelial cells, dynamic platelet-endothelial interactions (e.g., activation, aggregation, adhesion, translocation, and embolization) in the lumen that occur over large spatiotemporal scales, and often do not incorporate perfusion of whole blood.

However, this type of lung-on-a-chip model still lacks relevant functionality for mimicking relevant foundational conditions of pulmonary thrombosis.

Based on this limitation, the device is not appropriate for perfusing whole blood and for studying blood cell-endothelial interactions.

Another limitation of the long-on-a-chip model is that it uses non-primary epithelial cell lines, A549 or NCI-H441.

Although this type of model mimics certain aspects of human lung function, it is not ideal in the context of mimicking physiologically-relevant hemostasis and thrombosis, as they are derived from tumors and, therefore, can potentially alter endothelial and platelet function.

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