Name: in vitro and in vivo model to study bacterial adhesion to the vessel wall under flow conditions
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This work was supported by the Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen G0466.10, 11I0113N; &#8220;Eddy Merckx Research Grant&#8221; and the &#8220;Sporta research Grant&#8221; for Pediatric Cardiology, UZ Leuven, Belgium (J.C.); the Center for Molecular and Vascular Biology is supported by the Programmafinanciering KU Leuven (PF/10/014), by the &#8220;Geconcentreerde Onderzoeksacties&#8221; (GOA 2009/13) from the University of Leuven and a research grant from Boehringer-Ingelheim.

Animal experiments were approved by the Ethical Committee of the KU Leuven.
1. Preparing Bacteria for&#160;In Vitro Perfusions and In Vivo Experiments
<ol>
	<li>We used S. aureus strain Newman for all experiments described in this manuscript. S. aureus Newman was stored in Brain Heart Infusion (BHI) with 10% glycerol at -80 &#176;C.</li>
	<li>Use a sterile loop to scrape the frozen bacteria off and inoculate in 5 ml Tryptic Soy Broth (TSB) O/N at 37 &#176;C (OD600 &#62;3).</li>
	<li>Wash the bacteria by centrifugation (2,600 g, RT, 5 min) and resuspend the bacterial pellet in 5 ml PBS (phosphate buffered saline).</li>
	<li>Prepare a 1 mg/ml solution of 5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester (carboxy-fluorescein) in ethanol. Dilute the 1 mg/ml carboxy-fluorescein solution to 150 &#181;g/ml in laboratory grade water (e.g., MilliQ water). Protect the tubes from light with aluminum foil and store at -20 &#176;C.</li>
	<li>Centrifuge the bacteria (2,600 x g, RT, 5 min). Resuspend the bacterial pellet in 800 &#181;l PBS and add 200 &#181;l (final concentration of 30 &#181;g/ml for perfusion experiments) or 400 &#181;l (final concentration 50 &#181;g/ml for in vivo experiments) of the 150 &#181;g/ml carboxy-fluorescein solution. Protect the tubes from light with aluminum foil and incubate for 30 min at RT on a shaker.</li>
	<li>After labeling, block with 6% bovine serum albumin (BSA) solution in PBS.</li>
	<li>Dilute bacteria using optical densitometry (OD), an OD600 of 0.65 for in vitro experiments (corresponding to approximately 3 x 108 colony forming units (CFU)/ml for S. aureus) and an OD600 of 1.8 for in vivo experiments (corresponding to approximately 1 x 109 CFU/ml for S. aureus) in PBS. Protect the tubes from light with aluminum foil and leave on ice.</li>
</ol>
2. In Vitro Perfusion Experiments
<ol>
	<li>Coating of Glass Coverslips
	<ol>
		<li>Dilute von Willebrand factor (VWF) (Haemate P, stock concentration 2,400 &#181;g/ml) in laboratory grade water (deionized distilled) to a final concentration of 50 &#181;g/ml.</li>
		<li>Dilute collagen in isotonic glucose solution (SKF solution, pH 2.7-2.9, as supplied by the manufacturer) to a final concentration of 160 &#181;g/ml.</li>
		<li>Coat glass coverslips (24 &#215; 50 mm) with VWF or collagen by dropping 200 &#181;l of the coating on parafilm and place the coverslip on top of the droplet. The droplet will spread along the surface of the coverslip.</li>
		<li>Incubate the coverslip in a humidified container for 4 hr at RT. Carefully lift the coverslips from the parafilm with a blunt needle. Mount the coverslip in the bottom part of the flow chamber.</li>
	</ol>
	</li>
	<li>Coating of Plastic Slips with Endothelial Cells
	<ol>
		<li>Coat plastic slips (1-well PCA cell culture chamber, Sarstedt, Germany) with 1 ml of a 1% gelatin solution in PBS and incubate for 30 min at 37&#176;C. Seed human umbilical vein endothelial cells (HUVECs) on the gelatin coated plastic slips and grow them to 70-80% confluency. Mount the plastic slip in the bottom part of the flow chamber.</li>
	</ol>
	</li>
	<li>Perfusion Experiment 
	NOTE: A schematic overview of the in vitro perfusion model is represented in Figure 1.
	<ol>
		<li>Perform in vitro bacterial adhesion studies in a micro-parallel plate flow chamber at a laminar shear stress between 2.5 dyne/cm2 and 20 dyne/cm2 to simulate different physiological flow conditions.</li>
		<li>The flow chamber (in-house design) consists of a metal frame and a perfusion chamber made out of plexiglas (poly(methyl) methacrylate (PMMA)). By connecting it to a high-accuracy infusion pump (PHD 2000 Infusion, Harvard Apparatus, USA), we can generate flow rates between 0.0001 &#181;l/min and 220.82 ml/min.</li>
		<li>Connect the tubing to the upper part of the flow chamber and inject medium in the tubing. Gently place the upper part of the flow chamber on top of the bottom part and assemble the flow chamber. Be careful to avoid air bubbles. Inject 1 ml of medium through the chamber to make sure that the chamber is not leaking and to remove excess coating solution. Avoid air bubbles.</li>
		<li>Place the mouse on a thermo-controlled heating pad at 37 &#176;C on a microscope tray. Since this is a terminal procedure, there is no need for strict asceptic procedures. Make an incision near the jugular vein, gently remove the right side of the cervical muscle and isolate the jugular vein from the surrounding tissue.
		</li>
		<li>Set up the infusion pump and fluorescence microscope. Infusion pump settings depend on the syringe diameter and the desired flow rate (See section 2.4). From now on, work in a dark room.</li>
		<li>VWF Coating:
		<ol>
			<li>Fill a syringe with fluorescently labeled bacteria and connect it to the inlet tube. Avoid air bubbles. Start the infusion pump for 10 min. The infusion time depends on the shear rate and the coating, bacteria and medium used and should represent the steady state of adhesion.</li>
			<li>After 10 min, wash away unbound bacteria by connecting a syringe with PBS to the inlet tube and starting the infusion pump.</li>
			<li>Take at least 15 images or movies at different locations after the wash process. Bacteria are small and potentially difficult to focus on. Prior to the in vitro flow experiment, the appropriate focal plane can be retrieved by placing a drop of fluorescently labeled bacteria on a coverslip and placing the coverslip in the flow chamber. Then, search for the appropriate focal plane and save the settings. 
			NOTE: During the in vitro flow experiment, capturing the images during the washing step (&#177; 5 min after start) ensures that only the signal for adherent bacteria is captured.</li>
		</ol>
		</li>
		<li>Collagen Coating:
		<ol>
			<li>Add 60 &#181;g/ml VWF to the fluorescently labeled bacteria just before starting the perfusion. Fill a syringe with fluorescently labeled bacteria or fluorescently labeled bacteria supplemented with 60 &#181;g/ml VWF and connect it to the inlet tube. Avoid air bubbles. Repeat steps 2.3.5.2 to 2.3.5.3</li>
		</ol>
		</li>
		<li>Endothelial Cells:
		<ol>
			<li>Activate the endothelial cells by perfusion with a 0.1 mM solution of the Ca2+-ionophore A23187 (stock solution 10 mM dissolved in dimethyl sulfoxide (DMSO)) in DMEM at the same shear rate as the bacterial perfusion for 10 min by perfusion with a 0.1 mM. Repeat steps 2.3.5.2 to 2.3.5.3.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calculate shear rate and shear stress as follows. 
	Shear rate = 6Q/wh2 
	Where: Q: flow rate in ml/min, w: width in cm, h: height in cm 
	Shear stress (&#964;) = shear rate x viscosity (&#181;) 
	Where &#181;: medium: 0.01 dynes x sec/cm2 , whole blood: 0.04 dynes x sec/cm2 
	&#160;</li>
	<li>Image analysis
	<ol>
		<li>Obtain live images using an inverted fluorescence microscope with a black and white camera and develop using imaging software. Use exposure time of 1.5 sec. Take multiple snapshots (at least 15) randomly spread over the coated surface of the flow chamber and save them in the appropriate file format.</li>
		<li>Perform image analysis with ImageJ. Subtract the background to remove smooth continuous backgrounds from the image (Process &#8211; Substract Background) and define the threshold to set lower and upper threshold values, segmenting gray scale images into features of interest. Measure the area limited to the threshold.</li>
		<li>Compare bacterial adhesion, expressed as fluorescent area, e.g., using statistical analysis software. Compare the groups using one-way ANOVA or two-tailed Student&#8217;s t- test. Report all values as mean &#177; standard error of the mean (SEM). Consider a p-value of &#60;0.05 significant (* p &#60;0.05; ** p &#60;0.01; *** p &#60;0.001).</li>
	</ol>
	</li>
</ol>
3. In Vivo Mesenteric Perfusion Model
<ol>
	<li>Preparation/surgery of the mouse
	<ol>
		<li>Fast the mouse the night before the experiment in order to limit bowel movement.</li>
		<li>Give a 6-8 week old mouse (C57Bl/6) pre-operative analgesia by a subcutaneous injection of buprenorphine (0.1 mg/kg body weight (BW)) 20-30 min&#160;prior to the surgery.</li>
		<li>Anesthetize the mouse by intra-peritoneal injection of ketamine (125 mg/kg BW) and xylazine (12.5 mg/kg BW). Check by pedal reflex. Apply vet ointment to prevent dryness.</li>
		<li>Place the mouse on a thermo-controlled heating pad at 37 &#176;C on a microscope tray. Since this is a terminal procedure, there is no need for strict asceptic procedures. Make an incision near the jugular vein, gently remove the right side of the cervical muscle and isolate the jugular vein from the surrounding tissue. &#160;</li>
		<li>Insert a 2 French intravenous catheter into the right jugular vein for infusion of fluorescently labeled bacteria or other solutions. Open the peritoneal cavity via a midline abdominal incision and use cotton swabs to spread the mesenterium and to visualize the mesenteric arteriolar and venular circulation.</li>
		<li>Place the mouse on the right side on a transparent plate and secure the cannula with tape. Use a hot pack to prevent hypothermia. To prevent dehydration of the tissue, drop 500 &#181;l 0.9 % NaCl on the intestines.</li>
	</ol>
	</li>
	<li>Fluorescence microscopy of bacterial adhesion to the mesenteric circulation
	<ol>
		<li>Work in a dark room. Use cotton swabs to immobilize the vessels and visualize them under an inverted microscope.</li>
		<li>Topically apply 5 &#181;l of a 10 mM solution of the Ca2+-ionophore A23187 dissolved in DMSO. After 10 sec, inject 100 &#181;l labeled bacteria (see step 1) through the jugular catheter. Take time-lapse images.&#160;After the experiment is finished, euthanize the mouse according to institutional approved guidelines.</li>
	</ol>
	</li>
	<li>Image Analysis
	<ol>
		<li>Obtain live images using an inverted fluorescence microscope, captured using a black and white camera and developed using any imaging software. Apply automated exposure time and contrast optimization specific to the equipment used.</li>
		<li>Acquire time-lapse images using the &#8216;Acquisition&#8217; tool in the toolbar (Multidimensional Acquisitions &#8211; Time) using 40 cycles of 1,000 images/sec. Save the images in an appropriate image file format.</li>
		<li>Process images using ImageJ analysis software to measure the area of fluorescent signal per image. Define the threshold to set lower and upper threshold values, segmenting gray scale images into features of interest. Identify the region of interest (blood vessel) and measure the area limited to the threshold and the region of interest. Compare bacterial adhesion, expressed as fluorescent area using any statistical or graphing software.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Shear stress is a crucial factor for the early bacterial adhesion to the vessel wall and for the subsequent generation of endovascular or endocardial vegetations and metastatic infections4,5. We described complementary in vitro and in vivo models to study the pathogenesis of endovascular infections under physiological shear stress. These models have allowed us to identify von Willebrand factor-binding protein (vWbp) as the major S. aureus protein to interact under flow with an injured vascular wall exposing VWF4.
Endovascular infections, and infective endocarditis in particular, are of concern not only because of sepsis-induced organ failure and death, but also because of local and distant (&#8216;metastatic&#8217;) complications. To cause infective endocarditis and metastatic infections, bacteria have to adhere to the vessel wall and thus resist the shear stress of flowing blood. Most studies on bacteria virulence factors have been performed in static conditions. However, these established interactions might not withstand shear forces and studies under flow conditions can reveal new, previously unrecognized factors in bacteria-host interplay.
Using the micro-parallel flow chamber, we and others have shown the importance of VWF for vascular adhesion. Under shear stress, VWF progressively unfolds from its resting globular structure, and exposes the A1 domain that interacts with platelets via its GPIb receptor6. Flow chambers have been extensively used to study platelet function7.
Remarkably, also S. aureus adhesion under flow requires VWF, and in particular the A1 domain that is exposed upon shear. We identified vWbp to mediate VWF binding. vWbp is a coagulase that contributes to S. aureus pathophysiology by activating the host&#8217;s prothrombin. Staphylothrombin, the resulting complex of a bacterial coagulase and prothrombin, converts fibrinogen into insoluble fibrin8,9. Our studies have shown that vWbp does not only activate prothrombin, but triggers the formation of bacteria-fibrin-platelet aggregates, which enhance the adhesion to blood vessels under flow4,10,11.
The in vitro flow chamber model allows to study the different players in bacterial adhesion to cellular or matrix components. Bacterial virulence factors can be studied by using mutants or innocuous bacteria expressing specific surface proteins. Alternatively, pharmacologic inhibitors or blocking antibodies can be added to the medium in the flow chamber. The role of host factors such as different constituents of extracellular matrix can be studied by using coverslips with different coatings. The coverslips can also be covered with endothelial cells, of which the activation status can be modulated by adding specific stimulators. Apart from the vascular wall, the contribution of host blood cells and plasma proteins can be studied by adding these factors to the flowing medium. Thus, different conditions of increasing complexity can be studied under standardized conditions of laminar flow to unravel the interactions that allow bacteria to adhere to the vessel wall in vivo.
Interactions identified in the in vitro model are subsequently studied in an animal model to test their relevance in a complex organism. Other in vivo models to study dynamic interactions under flow have been described, such as the hamster dorsal skinfold chamber12 and the cremaster model13. In comparison, the mesenteric perfusion model described here offers several advantages because of its ease of use, the possibility to vary host genetic background of the mice and to evaluate pharmacological interventions.
In conclusion, the described models offer the possibility to study surface proteins not only of S. aureus, but of many other microorganisms in different host backgrounds, to better understand the pathogenesis of vascular infections.

To establish endovascular infections, pathogens require a mechanism to adhere to the endothelium, which lines the vessel wall and the inner surface of the heart, and to persist and establish an infection despite being exposed to the shear stress of rapidly flowing blood. The most frequent pathogen causing life-threatening endovascular infections and infective endocarditis is Staphylococcus aureus (S. aureus)1. 
Various bacterial surface-bound adhesive molecules mediate adhesion to host tissue by interacting with extracellular matrix components. These MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) recognize molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF). MSCRAMMs are important virulence factors of S. aureus and are implicated in the colonization and invasion of the host2. Most studies on these virulence factors have been performed in static conditions, and thus may not be representative for human infections where initial adhesion of the bacteria occurs in flowing blood. 
In the case of bloodstream infections, bacteria need to overcome the shearing forces of flowing blood in order to attach to the vessel wall. Models that investigate the interaction between bacteria and endothelium or subendothelium under flow conditions are therefore of particular interest. 
A recent study showed that the adhesion of S. aureus to blood vessels under shear stress is mediated by VWF3. VWF, a shear stress-operational protein, is released from endothelial cells upon activation. Circulating VWF binds to collagen fibers of the exposed subendothelial matrix. Our group reported that the von Willebrand factor-binding protein (vWbp) of S. aureus is crucial for shear-mediated adhesion to VWF4. 
In this article, we present an in vitro flow chamber model where bacterial adhesion to different components of the extracellular matrix or to endothelial cells can be evaluated. To validate the findings from in vitro data, we have developed an in vivo model that visualizes and quantifies the direct interaction of bacteria with the vessel wall and the formation of bacteria-platelet thrombi in the mesenteric circulation of mice, using real-time intravital vascular microscopy.

<table><tbody><tr><td>Brain Heart Infusion (BHI)</td><td>BD Plastipak&amp;nbsp;</td><td>237500</td><td></td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>Oxoid</td><td>CM0129</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Invitrogen</td><td>14190-169</td><td>D-PBS</td></tr><tr><td>5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester&amp;nbsp;</td><td>Sigma-Aldrich</td><td>21878-25MG-F</td><td>fluorescent labeling&amp;nbsp;</td></tr><tr><td>Bovine Serum Albumin Fraction V (BSA)</td><td>Roch</td><td>10 735 086 001</td><td></td></tr><tr><td>Haemate-P</td><td>CSL Behring</td><td>PL 15036/0010</td><td>VWF</td></tr><tr><td>Horm collagen</td><td>Takeda</td><td>10500</td><td>collagen</td></tr><tr><td>1-well PCA cell culture chambers</td><td>Sarstedt</td><td>94.6140.102</td><td>plastic slips</td></tr><tr><td>Temgesic</td><td>Reckitt Benckiser</td><td>283716</td><td>bruprenorphine</td></tr><tr><td>Anesketin (Ketamin hydrochloride 115 mg/ml (100 mg/ml ketaminum))</td><td>Eurovet</td><td>BE-V136516</td><td>ketamin</td></tr><tr><td>XYL-M 2% (xylazine hydrochloride 23.32 mg/ml (20 mg/ml xylazine))</td><td>VMD Arendonk</td><td>BE-V170581</td><td>xylazine</td></tr><tr><td>2 french intravenous catheter green</td><td>Portex</td><td>200/300/010</td><td></td></tr><tr><td>0,9% Sodium chloride (NaCl)</td><td>Baxter Healthcare</td><td>W7124</td><td></td></tr><tr><td>cotton swabs</td><td>International Medical Product</td><td>300230</td><td></td></tr><tr><td>Ca2+-ionophore solution A23187</td><td>Sigma-Aldrich</td><td>C7522-10 MG</td><td></td></tr><tr><td>26 gauge 1 ml syringe&amp;nbsp;&amp;nbsp;</td><td>BD Plastipak&amp;nbsp;</td><td>300013</td><td></td></tr><tr><td>26 gauge 1 ml syringe&amp;nbsp; with needle</td><td>BD Plastipak&amp;nbsp;</td><td>300015</td><td>intra-peritoneal injection</td></tr><tr><td>Centrifuge 5810-R</td><td>Eppendorf</td><td>5811 000.320</td><td></td></tr><tr><td>Glass cover slips (24x50)</td><td>VWR</td><td>BB02405A11</td><td>Thickness No, 1</td></tr><tr><td>PHD 2000 Infusion</td><td>Harvard Apparatus</td><td>702100</td><td>High-accuracy Harvard infusion pump</td></tr><tr><td>Axio-observer DI</td><td>Carl-Zeiss</td><td>Inverted fluorescence microscope</td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health</td><td>Analysis software</td><td></td></tr><tr><td>Graphpad Prism 5,0</td><td>Graphpad Software</td><td>Analysis software</td><td></td></tr><tr><td>AxioCam MRm</td><td>Carl-Zeiss&amp;nbsp;</td><td>Black and white camera</td><td></td></tr></tbody></table>

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 shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

S. aureus adhesion to VWF, subendothelial matrix and endothelial cells is a shear stress dependent phenomenon
To emphasize the role of shear stress in the interaction between S. aureus and VWF, we performed perfusions over VWF coated coverslips at different shear rates (a schematic overview of the in vitro perfusion model is given in Figure 1. Adhesion of S. aureus to VWF increased with increasing shear rates from 250 sec-1 to 2,000 sec-1 (Figure 2), indicating that high shear forces do not inhibit but reinforce the adhesion of bacteria to VWF.
In order to investigate the contribution of VWF to bacterial adhesion to collagen, the main component of the subendothelial matrix, we perfused fluorescently labeled S. aureus over collagen in the presence or absence of VWF. In the absence of VWF, adhesion of S. aureus to collagen decreased with increasing shear rates. However, when VWF was present in the medium, the adhesion of S. aureus increased with increasing shear rates (Figure 3).
The in vitro flow model also allows us to examine the adhesion of bacteria to endothelial cells under flow. We perfused HUVECs with fluorescently labeled S. aureus at shear rates from 500 to 2,000 sec-1. Where indicated, HUVECs were activated with a Ca2+-ionophore, to cause release of VWF. Endothelial cell activation and the subsequent VWF release, increased adhesion of S. aureus (Figure 4A), which formed typical &#8220;string-like&#8221; patterns of fluorescently labeled bacterial clusters aligned in the direction of the shear force (Figure 4B), suggesting the binding of bacteria along a linear-stretched VWF molecule.
Initial in vivo bacterial adhesion in splanchnic veins is mediated by VWF
Since S. aureus is able to adhere to VWF, we used wildtype mice (Vwf+/+) and VWF-deficient mice (Vwf&#8722;/&#8722;) to investigate bacterial adhesion to the activated vessel wall in vivo. Real-time videomicroscopy of splanchnic veins allowed the in vivo visualization of circulating fluorescently labeled S. aureus (Schematic overview of the in vivo perfusion model is represented in Figure 5).
After pharmacological activation of the endothelium by the Ca2+-ionophore, we observed rapid local accumulation of individual bacteria and aggregates of bacteria to the vessel wall of WT mice (supplemental Videos 1 and 2). Almost no adhesion of bacteria was observed on the activated vessel wall of Vwf-deficient mice (supplemental Video 3) compared with adhesion in WT mice (Figure 6). The absence of VWF decreases the ability of S. aureus to adhere to the activated vessel wall.
<img alt="Figure 1" src="/files/ftp_upload/52862/52862fig1.jpg" /> 
Figure 1.&#160;A schematic representation of the in vitro flow model. The in vitro flow model is a multifunctional model, which allows the study of different shear dependent mechanisms such as bacterial adhesion to the subendothelial matrix but also thrombus formation. The micro-parallel flow chamber is placed on a coverslip (plastic or glass) with different coatings of proteins and endothelial cells. The adhesion of different bacteria (orange and grey dots) can be analyzed, and the impact of the presence of plasma proteins, platelets and whole blood can be evaluated. Fluorescent markers for platelets (blue ovals) or fibrinogen (blue strings) can be used in combination with different inhibitors (black ovals) to distinguish bacterial and host factors. Representative images of bacterial adhesion of S. aureus to collagen coating in the presence (bottom) or absence (top) of VWF are shown (scale bar is 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52862/52862fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52862/52862fig2.jpg" /> 
Figure 2.&#160;Adhesion of S. aureus to VWF increases with increasing shear rates. Micro-parallel flow chamber perfusion over coated VWF (50 &#181;g/ml) with fluorescently labeled S. aureus Newman at shear rates of 250 to 2,000 sec-1 (sec-1) in medium (n &#62;5). All results are expressed as mean &#177; SEM. *p &#60;0.05, **p &#60;0.01.
<img alt="Figure 3" src="/files/ftp_upload/52862/52862fig3.jpg" /> 
Figure 3.&#160;Adhesion of S. aureus to subendothelium is shear and VWF dependent. Micro-parallel flow chamber perfusion over coated collagen (160 &#181;g/ml) with fluorescently labeled S. aureus Newman at shear rates of 250 to 2,000 sec-1 in medium (n &#62;5). VWF (60 &#181;g/ml) was present in the medium where indicated. All results are expressed as mean &#177; SEM. **p &#60;0.01.
<img alt="Figure 4" src="/files/ftp_upload/52862/52862fig4.jpg" /> 
Figure 4.&#160;Adhesion of S. aureus to activated endothelial cells is shear dependent. Micro-parallel flow chamber perfusion over endothelial cells. (A) Human umbilical vein endothelial cells were activated with the Ca2+-ionophore A23187 (0.1 mM) followed by a 10 min perfusion of fluorescently labeled S. aureus Newman at shear rates of 500 to 2,000 sec-1 in medium (n &#62;5). All results are expressed as mean &#177; SEM. *p &#60;0.05. (B) Image of micro-parallel flow chamber perfusion over activated HUVECs with S. aureus at a shear rate of 1,000 sec-2. S. aureus forms strings of &#177; 200 microns length, suggesting adhesion to VWF multimers (scale bar is 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52862/52862fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52862/52862fig5.jpg" /> 
Figure 5.&#160;A schematic overview of the in vivo mesenteric perfusion model. A right jugular vein catheter (yellow line) is inserted for the administration of fluorescently labeled bacteria (orange dots), additional anesthetics or other components such as pharmaceutical inhibitors and antibodies. The peritoneal cavity is opened and the mesenterium is spread to visualize the blood vessels (venous and arterial) under a fluorescence microscope. After pharmacological activation of the endothelium by a Ca2+-ionophore, which induces the release of VWF, bacteria can be injected through the jugular vein catheter. Real-time intravascular video microscopy allows the in vivo visualization of circulating fluorescently labeled bacteria and the resulting formation of bacteria-platelet thrombi. <a href="https://www.jove.com/files/ftp_upload/52862/52862fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52862/52862fig6.jpg" /> 
Figure 6.&#160;The initial adhesion of S. aureus to activated endothelium in vivo is mediated by VWF. In vivo venous mesenteric perfusion model with C57Bl/6-Vwf+/+ and C57Bl/6-Vwf-/- mice. Adhesion of fluorescently labeled S. aureus to the locally activated vessel wall is significantly lower in Vwf-/- mice. All results are expressed as mean &#177; SEM. ***p &#60;0.001, n &#62;7.
<img alt="Video Figure 1" src="/files/ftp_upload/52862/52862figV1.jpg" /> Video 1: Real-time adhesion of S. aureus to activated vessel wall in Vwf+/+ mice. <a href="https://www.jove.com/files/ftp_upload/52862/Video1JorienClaes.mov" target="_blank">Please click here to view this video.</a>
<img alt="Video Figure 2" src="/files/ftp_upload/52862/52862figV2.jpg" /> Video 2: Real-time aggregate formation and embolization of S. aureus in Vwf+/+ mice. <a href="https://www.jove.com/files/ftp_upload/52862/Video2JorienClaes.mov" target="_blank">Please click here to view this video.</a>
<img alt="Video Figure 3" src="/files/ftp_upload/52862/52862figV3.jpg" /> Video 3: Real-time adhesion of S. aureus to activated vessel wall in Vwf-/- mice. In vivo mesenteric perfusion model with Vwf+/+ and Vwf-/- mice. Five &#181;l of a Ca2+-ionophore (10 mM) was applied to the region of the visualized vascular bed. A suspension of carboxy-fluorescein-labeled S. aureus was injected through the jugular catheter. The mesenteric circulation was visualized under an inverted microscope. <a href="https://www.jove.com/files/ftp_upload/52862/Video3JorienClaes.mov" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions at JoVE.com

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

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.6K Views.  KU Leuven. To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion. 

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

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Bioengineering

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Developmental Biology

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

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.

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 ...

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

Summary

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Chapters in this video

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Introducing Shear Stress in the Study of Bacterial Adhesion

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

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