Name: in vitro microfluidic disease model to study whole blood-endothelial interactions and blood clot dynamics in real-time
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We thank Jan Voorberg from the Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands, for his input in this manuscript. This work was supported by the Dutch CardioVascular Alliance (DCVA) [2012-08, 2014-11] awarded to the Phaedra and the Reconnect consortium as well as the Impulse grant 2018 awarded to the Phaedra IMPACT consortium. These grants include collective funding by the Dutch Heart Foundation, Dutch Federation of University Medical Centers, The Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences. Furthermore, this work was funded by the European Research Council under the Advanced Grant &#8216;VESCEL&#8217; program (Grant number: 669768). XDM is funded by a research grant of the Institute for CardioVascular Research (ICaR-VU) at the VU University Medical Center, Amsterdam, the Netherlands.

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The authors declare no conflict of interest.

Coagulation is a result of the complex and temporally controlled interplay between the endothelium and blood components. This in vitro assay presents a method to investigate thrombogenic properties of endothelial cells in real-time. Various types of primary human endothelial cells can be used, facilitating in situ thrombosis in an organ and patient-specific&#160;manner. In this study, we illustrated the use of this protocol comparing thrombogenic properties of PAECs isolated from healthy donors versus CTEPH patients. Live thrombus formation was studied by perfusion of whole blood from healthy subjects over&#160;histamine-activated endothelium, while the effect of a DOAC was tested as an antithrombotic agent.
Besides the use of commercially available microchannels, as shown in the representative results, the introduction of the custom-made microfluidic channels enables the study of the influence of vascular geometry changes on thrombus formation. For example, flow decreases at a branch point or stenosis results in an increase in shear stress and more platelet activation36. However, a significant limitation of these custom-made microfluidic channels is the requirement of high cell numbers to form a stable monolayer of ECs as described in step 2.3.2. This may present&#160;a limiting factor when patient-derived cells are scarce. The strengths of the commercially available microslides are that surface areas are modified for cell culture and growth areas are bigger, thereby allowing ECs to form a confluent and stable monolayer. On the other hand, a bigger surface area will result in a bigger lumen. According to Equation 1, this requires higher blood volumes to reach similar flow rates as in the custom-made microfluidics.
An improvement to&#160;this protocol could be to investigate live EC&#160;loss or changes in barrier integrity. EC&#160;damage in this protocol is only measured at the end of the experiment. For live tracking, ECs can be tagged with mCherry VE-cadherin, for example37. However, as this would need a highly optimized protocol with efficient virus transfection, Electric Cell-substrate Impedance Sensing (ECIS) could be used as an alternative to study endothelial integrity and barrier function38. Perfusion over special ECIS flow channels allows longitudinal monitoring of endothelial barrier integrity under flow. These specific ECIS features allow for parallel measurements of endothelial barrier properties and thrombus formation. Alternative ways for parallel EC barrier measurements, especially in the custom-made arrays include the use of fluorescent dextrans in the perfusate, which diffuse out of the lumen, depending on the EC barrier properties.
A limitation of the described protocol is that ECs are removed from the human body and cultured on tissue culture plastic, which is a stiff, artificial substrate. Cells adapt to their biophysical environment. This could possibly affect endothelial response to platelet activation, as there is an association between platelet activation and wall stiffness39. Despite these adaptations to culture plastics, cells do&#160;keep disease-specific characteristics that can be identified in direct comparison with ECs derived from healthy donors as shown with&#160;control, CTEPH-PAEC, and HUVEC that exerted&#160;different patterns of platelet adhesion and fibrin deposition after 5 min of blood perfusion.
In contrast to other protocols, this system uses whole blood while others&#160;study EC interaction with a single blood component such as platelets and leukocytes19,20,21. There have been more advanced microfluidic models developed that allow the study of endothelial function in a vascular model with a round vessel geometry and soft extracellular matrix. However, these are optimized with HUVECs40,41,42. The novelty of the described protocol is the use of primary ECs combined with whole blood&#160;bringing the modelling of in situ thrombosis one step closer to in vivo conditions. Having optimized the protocol for the use of patient-derived ECs and patient-derived blood further optimizes disease modelling in vitro, allowing the assessment of personalized thrombus formation and drug treatment.
The described protocol can be applied to study the effect of anticoagulation therapies on patient-derived cells. While we used dabigatran to inhibit&#160;thrombus formation, it is possible to use other&#160;anticoagulants, such as rivaroxaban, which directly inhibits factor Xa in the coagulation cascade. Direct-platelet inhibitors like clopidogrel or aspirin can be studied as well, as these act on the primary phase of hemostasis where platelets bind to ECs. Ultimately, the thrombogenic capacities of patient-specific ECs in interaction with the patient&#39;s own blood can be used to predict the personal effect of anticoagulation therapy on the individual patient. Furthermore, a knockdown of specific proteins can provide additional functional information.
There are some steps in the described protocol that are critical for a successful perfusion experiment. First, during the isolation of primary cells, it is necessary to obtain a highly pure EC culture. Second, it is important that the ECs form a stable confluent monolayer. If this is not the case, a slight change in shear stress can cause endothelial damage and activation of the coagulation cascade, or platelets can start binding to the basement membrane, which will provide false positive results. Third, it is essential to prevent air bubbles, as those can damage the endothelium and thereby influence the results.
After recalcification of the citrated blood with calcium chloride and magnesium chloride, it is important to immediately start the perfusion experiment. Recalcification induces a rapid response in platelet activation and thrombus formation, resulting in fast clotting in the sample.
In conclusion, we describe a highly versatile protocol to study whole blood-endothelial cell interactions during thrombosis.&#160;

The endothelium forms the inner cellular layer of blood vessels and separates blood from the surrounding tissue. It has been described as a dynamic organ that actively regulates its micro-environment and responds to external stimuli1. Because of its direct contact with flowing blood, the endothelium is pivotal in the control of hemostasis and thrombosis and is the primary source of many of the major regulatory molecules that control platelet aggregation, coagulation, and fibrinolysis2. Healthy, nonactivated endothelial cells (EC) produce several molecules that counteract platelet activation and prevent coagulation and thrombus formation to maintain blood flow, such as prostacyclin, thrombomodulin, or tissue factor pathway inhibitor (TFPI)2,3. This prevents the adhesion of platelets, platelet aggregation, and thrombus formation. Injury or activation of the vessel wall results in a procoagulant endothelial phenotype that initiates localized platelet adhesion and clot formation2,4. Upon endothelial activation platelets&#160;adhere to von Willebrand Factor (VWF), a multimeric protein released from ECs, or to exposed binding sites of the underlying subendothelial matrix. Subsequently, molecular changes in platelets and the exposure to tissue factor (TF) initiate the activation of the coagulation system, which induces thrombus formation by fibrin polymerization5,6. Together, the resulting clot provides the basis for wound closure by re-endothelialization7. Perturbations of the coagulation system may result in bleeding disorders, such as von Willebrand disease, hemophilia, or thrombosis, that often result from&#160;a dysregulated pro- and antithrombotic balance of the endothelial hemostatic pathway2,3.
The process of hemostasis occurs in both arterial and venous circulation. However, the mechanisms underlying arterial and venous thrombosis are fundamentally different. While arterial thrombosis, as seen in ischemic heart disease, is mostly driven by the rupture of an atherosclerotic plaque under conditions of high shear stress, venous thrombosis mostly develops in the absence of endothelial injury in a condition of stasis8,9,10. A deep vein thrombus may embolize and travel towards the pulmonary arteries, where it causes a pulmonary embolism. This can result in chronic vascular obstructions leading to significant impaired functional capacities&#160;that might foster the development of chonic lung diseases&#160;including the development of chronic thromboembolic pulmonary hypertension (CTEPH)11,12,13,14. CTEPH is characterized by elevated pulmonary pressure due to obstructions of the pulmonary arteries by thromboembolic material following at least three&#160;months of anticoagulation therapy15. In addition to lung emboli, it is postulated that the pulmonary endothelium provides a prothrombotic environment in CTEPH that facilitates&#160;in situ thrombosis and chronic obstructions of the pulmonary arteries, causing the increase in blood pressure that ultimately can result in heart failure, if untreated16,17.
Over the past years, various studies have led to the development of assays to examine thrombus formation by measuring platelet function and coagulation18. However, most of them either study the interaction of whole blood with single extracellular matrix components like collagens or fibrins, or endothelial function in interaction with single blood components, such as endothelial-platelet or endothelial-leukocyte interaction19,20,21,22. These assays are most commonly performed with human umbilical vein&#160;endothelial cells (HUVEC), as these cells are easily obtained. However, hemostatic genes are differentially expressed across the vascular tree, vessel types, and organ systems23,24, which makes the use of HUVECs to represent endothelial cells involved in arterial thrombosis or pulmonary embolisms problematic23.
In addition to EC plasticity, disease-specific hemodynamic alterations and changes in vascular morphology can promote thrombus formation at the normal endothelium25. Higher shear rates, due to local vasoconstriction or changes in vessel geometry, for example, may result in acute thrombus formation, causing a stenosis that accelerates the cessation of blood flow26. The use of custom-made microengineered flow channels allows to specifically design vascular geometries that are representative of the (patho)biology. In this way, it is possible to study the effect of local biomechanical forces on healthy or diseased EC27.
There are anticoagulation therapies available for targeting different phases and molecules in the coagulation cascade, which all pose&#160;particular risks and benefits that can be specific to certain disorders. The approach of disease modeling described in this paper is especially suited to test the effects of various anticoagulation and antiplatelet therapies on thrombus dynamics.
The aim is to present a model of thrombosis that includes primary ECs, yielding a versatile model suitable for the analysis of various forms of thrombosis depending on the type of primary ECs&#160;used. As an illustration, we used pulmonary artery endothelial cells from CTEPH patients in interaction with whole human blood containing all components involved in thrombus formation (platelets, leukocytes, erythrocytes, clotting proteins, and cofactors). This approach can be applied in commercial parallel flow channels or in custom-made microfluidic flow channels with a specific vascular design. As such, the model can eventually be used in the study of thrombus formation and resolution, for the assessment of inflammatory responses in disease modeling, for antiplatelet or anticoagulation therapy, and ultimately for personalized medicine.
This study describes the isolation of primary human pulmonary artery endothelial cells. For the isolation of other primary human endothelial cell types, we refer to previously published methods, including pulmonary microvascular endothelial cells25, human umbilical vein endothelial cells28, and blood circulating endothelial colony forming cells Figure 1A29.

<table border="0" cellpadding="0" cellspacing="0" style="width:895px;" width="894">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">20 mL syringe</td>
			<td style="width:200px;">BD Plastipak</td>
			<td style="width:161px;">300613</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">20X objective</td>
			<td style="width:200px;">Olympus</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2-Propanol (IPA)</td>
			<td style="width:200px;">Boom</td>
			<td style="width:161px;">76051455 . 5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Aladdin Syringe Pump</td>
			<td style="width:200px;">Word Precision Instruments</td>
			<td style="width:161px;">AL-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa488-Fibrinogen</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">F13191</td>
			<td>15 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa647 Goat anti Rabbit</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">A21245</td>
			<td align="right">0.180555556</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Biopsy punch 1 mm diameter, Integra Miltex</td>
			<td style="width:200px;">Ted Pella</td>
			<td style="width:161px;">15110-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">CaCl2</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">21115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Calcein AM-Red</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">C3099</td>
			<td>1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">CD42b-APC</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-100-208</td>
			<td>1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Citric Acid</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Collagen Typ I</td>
			<td style="width:200px;">Corning</td>
			<td>354249</td>
			<td>0,1 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Corning CellBIND Surface 60 mm Culture Dish</td>
			<td style="width:200px;">Corning</td>
			<td style="width:161px;">3295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Cross-flow hood</td>
			<td style="width:200px;">Basan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Desiccator</td>
			<td style="width:200px;">Duran</td>
			<td>24 782 69</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">D-Glucose</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">14431-43-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">EDTA</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">15575-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Endothelial Cell Medium (ECM)</td>
			<td style="width:200px;">ScienCell</td>
			<td style="width:161px;">1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fibronectin</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Flow tubings</td>
			<td style="width:200px;">ibidi</td>
			<td>10831, 10841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Gelatin</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">104070</td>
			<td>0.1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">HEPES</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Hoechst 33342</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">H1399</td>
			<td>1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ibidi &#181;-Slide VI 0.4 flow chambers</td>
			<td style="width:200px;">ibidi</td>
			<td style="width:161px;">80606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ImageJ</td>
			<td style="width:200px;">ImageJ</td>
			<td style="width:161px;">v1.49</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">KCl</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">7447-40-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lab oven</td>
			<td style="width:200px;">Quincy</td>
			<td>10GC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">LS720 Fluorescent microscope</td>
			<td style="width:200px;">etaluma</td>
			<td style="width:161px;">LS720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Luer connector</td>
			<td style="width:200px;">ibidi</td>
			<td style="width:161px;">10802, 10825</td>
			<td>Male elbow connectors, and Female tube connectors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">MACS magnetic beads (anti-CD144)</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-097-857</td>
			<td>1:5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">MgCl2</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microscopy slides, 76 x 26 mm</td>
			<td style="width:200px;">Thermo Scientific</td>
			<td style="width:161px;">AAAA000001##12E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Negative photoresist, SU-8</td>
			<td>Microchem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Non-Essential Amino Acids (NEAA)</td>
			<td style="width:200px;">Lonza</td>
			<td style="width:161px;">13-114E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Paraformaldehyde (PFA)</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">818715</td>
			<td>4% PFA in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Penicillin/streptomycin (P/S)</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:161px;">15140-122</td>
			<td>1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:161px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Plasma chamber, CUTE</td>
			<td style="width:200px;">Femto Science</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Polydimethylsiloxane (PDMS), Sylgard 184</td>
			<td style="width:200px;">Dow</td>
			<td>101697</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">sodium citrate blood collection tubes</td>
			<td style="width:200px;">BD Vacutainer</td>
			<td style="width:161px;">363048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">trisodium citrate</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">6448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:161px;">25300-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">VE-Cadherin (D87F2)-XP</td>
			<td style="width:200px;">Cell Signaling</td>
			<td style="width:161px;">2500</td>
			<td>1:300</td>
		</tr>
	</tbody>
</table>

in vitro microfluidic disease model to study whole blood-endothelial interactions and blood clot dynamics in real-time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

The representative results can be divided into three parts, each representing the respective steps of experimental setup, data collection, and data analysis. Depending on the research question, parameters for each step can be changed. The presented data are applied to study the influence of the&#160;endothelium on thrombus formation.
Experimental Setup 
It is well-established that endothelial cells are highly heterogeneous in structure and function, depending on location and time, during health and disease23. Various sources of endothelial cells can be used to study endothelial-blood interaction, which in this case were commercially available HUVECs and PAECs&#160;(Figure 1A). HUVECs have been the most commonly used in laboratory models, while PAECs are patient-derived isolated cells from the pulmonary arteries. Furthermore, there are&#160;well-established protocols available to isolate microvascular endothelial cells (MVEC) from the pulmonary circulation or blood circulating endothelial colony forming cells (ECFC)25,28,29.
After isolation, endothelial cells were characterized by VE-cadherin, CD31, and TIE2 staining to confirm an endothelial phenotype. Characterization for the presence of &#945;SMA and cytokeratin indicated the absence of a fibroblast or epithelial-like phenotype (Figure 1B). After obtaining a highly pure population of ECs, passage 3&#8211;5 cells were used to seed either a commercial microslide or custom-made microfluidic flow channels (Figure 1C). While the commercially available microslides are primarily parallel flow chambers, or Y-shaped channels with specifically defined parameters in height or bifurcation angle, the use of microfluidic slides makes it possible to adapt the experimental parameters to in vivo vessel geometry and blood flow dynamics31. However, custom-made microfluidic sizes are smaller&#160;and&#160;tend to limit&#160;cell spread and induce more cell death32,33. Using a surplus of cells compensates for the fact that only a small percentage of cells will adhere. This was observed when a stenosis was introduced, where endothelial cells showed a more elongated phenotype compared to a parallel microfluidic channel (Figure 1D). Commercial flow chambers have a bigger&#160;surface area that cells can bind to. This requires fewer cell numbers for seeding.
To study endothelial cell-blood interaction, whole blood was perfused over an endothelial monolayer. Citrated blood was collected on the day of the experiment and immediately before perfusion recalcified. Cells were stimulated with histamine to induce VWF release and platelet adhesion 30 min prior to perfusion (Figure 1E)34,35. Because of the small dimensions, the custom-made microfluidic channels allowed use of smaller blood volumes.
Data collection 
To investigate thrombus formation on PAECs, Calcein AM-Red fluorescently labeled blood cells and Alexa488-conjugated fibrin were perfused for 5 min at 2.5 dyne/cm2 (Figure 2A&#8211;C). Adherent blood cells and deposited fibrin were quantified. Images were acquired every 30 s and quantified with ImageJ. It was important to subtract the background to eliminate the autofluorescence of nonadhered platelets. The triangle algorithm for thresholding has been used to define minimal background. It allowed for measurement of small platelet aggregates (Figure 2D).
Expectedly, under nonstimulated conditions, there was no binding of platelets and fibrin to the endothelium. To promote VWF release and platelet binding, PAECs were stimulated with histamine, which resulted in an immediate increase of platelet adhesion reaching a plateau after 2.5 min. At this time, platelets started to secrete autocrine factors that induced platelet aggregation and fibrinogen cleavage into fibrin. Fibrin was deposited after 3 min and formed a stable aggregate with platelets after 4 min (Figure 2E).
To investigate whether this effect could be inhibited by a direct oral anticoagulant (DOAC), blood was treated with 10 nM dabigatran. Dabigatran was added to the blood dilution, where it inhibits&#160;Factor IIa in the coagulation pathway, and prevented the cleavage of fibrinogen to form fibrin fibers. When dabigatran-treated blood was perfused over stimulated PAECs, clot formation could be directly inhibited mainly by delaying fibrin deposition (Figure 2F).
Data analysis 
To study the influence of various endothelial sources on thrombus formation, the cellular changes upon 5 min&#160;blood perfusion were analyzed. The endothelium was fixed and adherent platelets were labeled with CD42b before imaging under a confocal microscope. This provided a&#160;detailed analysis for colocalization of platelets and fibrin that could indicate dysfunctional coagulation factors in the blood. The influence of EC&#160;in clot formation was determined by standard immunofluorescent staining. Endothelial cell-cell contacts were maintained, as confirmed by VE-cadherin staining,indicating that the blood clots formed on top of the&#160;endothelial monolayer rather than on the underlying matrix in between endothelial gaps (Figure 3). Furthermore, the use of different cell sources resulted in different patterns of thrombi&#160;forming&#160;on the endothelium. HUVECs are venous endothelial cells and showed limited&#160;platelet adhesion and fibrin deposition, while diseased primary PAEC from CTEPH patients showed abundant&#160;platelet adhesion and more fibrin deposition upon histamine stimulation compared to healthy PAEC. This suggests that the endothelium of CTEPH patients show higher responsiveness to vasoactivation that results in increased thrombus formation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61068/61068fig1.jpg" /> 
Figure 1: Schematic overview of the protocol. (A) Various sources and different types of endothelial cells were isolated and cultured for usage in a microfluidic flow channel for perfusion experiments. Representative brightfield images of different types of endothelial cells. Scale bar = 50 &#181;m. (B) Isolated cells were characterized by immunofluorescent staining to confirm an endothelial phenotype. Scale bar = 50 &#181;m. (C) Cells can be seeded in either commercial flow slides or custom-made microfluidic channel. (D) Representative brightfield images of HUVEC grown in different channel geometries. Scale bar = 50 &#181;m. (E) Experimental setup of blood perfusion experiments. Citrated blood was collected, diluted with saline buffer, and perfused over endothelial cells with a syringe pump. The lung and umbilical vein in this figure were modified from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic Licence. http://smart.servier.com/ <a href="https://www.jove.com/files/ftp_upload/61068/61068fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61068/61068fig2.jpg" /> 
Figure 2: Image acquisition and quantification of thrombus formation. (A) Representative time-lapse imaging of adhered Calcein AM-Red labeled platelets and deposited Alexa488-conjugated fibrin at 1, 3, and 5 min after whole blood perfusion over unstimulated PAECs (B) and over histamine stimulated PAECs. (C) Representative time-lapse images of platelet adhesion and fibrin deposition at 1, 3, and 5 min of perfusion with whole blood incubated with dabigatran perfused over histamine-stimulated PAECs. Scale bar = 50 &#181;m. (D) Schematic overview of image quantification in ImageJ. (E) Quantification of platelet adhesion and fibrin deposition for every 30 s on an unstimulated and histamine stimulated endothelium. (F) Quantification of the effect of dabigatran on thrombus formation quantified by platelet adhesion and fibrin deposition. Data are represented as mean &#177; SD, n = 3. <a href="https://www.jove.com/files/ftp_upload/61068/61068fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61068/61068fig3.jpg" /> 
Figure 3: Representative confocal images of flow experiments to characterize thrombus formation in platelet adhesion and fibrin deposition on endothelial cells. Endothelial cell-cell contacts were characterized by VE-cadherin and measured in control PAECs, patient-derived CTEPH PAECs, and HUVEC. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61068/61068fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time at JoVE.com

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

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An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...