Name: parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress
Uploaded: 2012-01-17T12:00:00
Duration: 12 min 43 s
Description: Watch this Scientific Journal Video about Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress at JoVE.com

The authors would like to thank Joe Owen in the Biomedical Instrument and Machine Shop for his tireless efforts in machining and assembling the flow chamber parts and Matt Maudsley from Leica Microsystems for assisting in techniques to image cells through flow chambers. We are indebted to Kevin Collins from Duke Perfusion Services and Dr. Steve Wallace in the Department of Biomedical Engineering for helpful suggestions on flow circuit design. We would also like to thank the National Science Foundation Graduate Research Fellowship Program for supporting Alexandra Jantzen and the NIH for their support through Grant "Autologous EPC lining to improve biocompatibility of circulatory assist devices" RC1HL099863-01.

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Heart Association Scientific Sessions, Abstract Oral Sessions, Medical Aspects End Stage Heart Failure: Transplantation and Device Therapies. , (2010).</li><li>Brown, M. A., Wallace, C. S., Angelos, M., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+umbilical+cord+blood-derived+late+outgrowth+endothelial+progenitor+cells+exposed+to+laminar+shear+stress.">Characterization of umbilical cord blood-derived late outgrowth endothelial progenitor cells exposed to laminar shear stress.</a> Tissue. Eng. Part. A. 15, 3575-3587 (2009).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+titanium+ventricular+assist+device+surfaces+after+gold/palladium+coating+for+scanning+electron+microscopy.">Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy.</a> Microsc. Res. Tech. 73, 71-76 (2010).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+titanium+cardiovascular+devices+seeded+with+autologous+blood-derived+endothelial+progenitor+cells:+EPC-seeded+antithrombotic+Ti+Implants.">The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants.</a> Biomaterials. 32, 10-18 (2011).</li><li>Rinker, K. D., Prabhakar, V., Truskey, G. 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A., Nevill, T., Ionescu-Zanetti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+Adhesion+and+Aggregation+Under+Flow+using+Microfluidic+Flow+Cells.">Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells.</a> J. Vis. Exp. (32), e1644-e1644 (2009).</li><li>Conant, C. G., Schwartz, M. A., Ionescu-Zanetti, C. W. e. l. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=plate-coupled+microfluidic+devices+designed+for+facile+image-based+cell+adhesion+and+transmigration+assays.">plate-coupled microfluidic devices designed for facile image-based cell adhesion and transmigration assays.</a> J. Biomol. Screen. 15, 102-106 (2010).</li><li>Conant, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+well-plate+microfluidic+devices+to+conduct+shear-based+thrombosis+assays.">Using well-plate microfluidic devices to conduct shear-based thrombosis assays.</a> J. Lab. Autom. 16, 148-152 (2011).</li><li>Metaxa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide-dependent+stimulation+of+endothelial+cell+proliferation+by+sustained+high+flow.">Nitric oxide-dependent stimulation of endothelial cell proliferation by sustained high flow.</a> Am. J. Physiol. Heart. Circ. Physiol. 295, H736-H742 (2008).</li><li>Radel, C., Carlile-Klusacek, M., Rizzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+caveolae+in+beta1+integrin-mediated+mechanotransduction.">Participation of caveolae in beta1 integrin-mediated mechanotransduction.</a> Biochem. Biophys. Res. Commun. 358, 626-631 (2007).</li><li>Wang, X. L., Fu, A., Spiro, C., Lee, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+Vascular+Endothelial+Cells-Effects+of+Laminar+Shear+Stress+and+High+Glucose.">Proteomic Analysis of Vascular Endothelial Cells-Effects of Laminar Shear Stress and High Glucose.</a> J. Proteomics. Bioinform. 2, 445-445 (2009).</li><li>Elfervig, M. K., Minchew, J. T., Francke, E., Tsuzaki, M., Banes, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1beta+sensitizes+intervertebral+disc+annulus+cells+to+fluid-induced+shear+stress.">IL-1beta sensitizes intervertebral disc annulus cells to fluid-induced shear stress.</a> J. Cell. Biochem. 82, 290-298 (2001).</li><li>Archambault, J. M., Elfervig-Wall, M. K., Tsuzaki, M., Herzog, W., Banes, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+tendon+cells+produce+MMP-3+in+response+to+fluid+flow+without+significant+calcium+transients.">Rabbit tendon cells produce MMP-3 in response to fluid flow without significant calcium transients.</a> J. Biomech. 35, 303-309 (2002).</li><li>Patton, J. T., Menter, D. G., Benson, D. M., Nicolson, G. L., McIntire, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computerized+analysis+of+tumor+cells+flowing+in+a+parallel+plate+chamber+to+determine+their+adhesion+stabilization+lag+time.">Computerized analysis of tumor cells flowing in a parallel plate chamber to determine their adhesion stabilization lag time.</a> Cell. Motil. Cytoskeleton. 26, 88-98 (1993).</li><li>Wiese, G., Barthel, S. R., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Physiologic+E-Selectin-Mediated+Leukocyte+Rolling+on+Microvascular+Endothelium.">Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium.</a> J. Vis. Exp. (24), e1009-e1009 (2009).</li><li>Kaper, H. J., Busscher, H. J., Norde, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+poly(ethylene+oxide)+brushes+on+glass+surfaces+and+adhesion+of+Staphylococcus+epidermidis.">Characterization of poly(ethylene oxide) brushes on glass surfaces and adhesion of Staphylococcus epidermidis.</a> J. Biomater. Sci. Polym. Ed. 14, 313-324 (2003).</li><li>Bakker, D. P., Plaats, A. v. a. n. d. e. r., Verkerke, G. J., Busscher, H. J., van der Mei, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+velocity+profiles+for+different+flow+chamber+designs+used+in+studies+of+microbial+adhesion+to+surfaces.">Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces.</a> Appl. Environ. Microbiol. 69, 6280-6287 (2003).</li><li>Angelos, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+adhesion+of+umbilical+cord+blood+endothelial+progenitor+cells+under+laminar+shear+stress.">Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress.</a> Biophys. J. 99, 3545-3554 (2010).</li><li>Baker, G. R., Sullam, P. M., Levin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+fluorescent+method+to+internally+label+platelets+suitable+for+physiological+measurements.">A simple, fluorescent method to internally label platelets suitable for physiological measurements.</a> Am. J. Hematol. 56, 17-25 (1997).</li></ol>

Production and Free Access of this video-article is sponsored by Cole-Parmer Instrument Co.

Our flow circuit and flow chamber allow us to subject adherent cells, e.g. EPCs, to defined fluid shear stresses. Since the chamber top and bottom are transparent, cell adhesion and morphology can be evaluated either in real-time, through the chamber itself, or after a flow experiment and disassembly of the flow chamber. At that point, cells can be harvested under sterile conditions and either re-plated, or used to collect their DNA or RNA, etc., for further analysis.
To achieve laminar flow, the design of the chamber must be such that several conditions are met.
First, the flow must be laminar, which can be verified by calculating its Reynolds number (Re), which is the ratio of inertial forces to viscous forces. (If viscous forces predominate, Re is small and the flow is laminar or 'fully developed' - usually for Re &lt; 2300. If inertial forces predominate, the flow becomes more and more random until it is turbulent, as is the case for Re &gt; 4000.) We can calculate Re according to equation 3 8,
<img alt="Equation 3" src="/files/ftp_upload/3349/3349eq3.jpg" />
Where &rho; is the fluid density, Q is the flow rate, &mu; is the viscosity, w and h are the width and height of the chamber, respectively, and Dh is the hydraulic diameter, defined according to equation 48,
<img alt="Equation 4" src="/files/ftp_upload/3349/3349eq4.jpg" />
The Reynolds number of our three flow chambers, with heights ranging from 166 - 267 &mu;m, range from 13.9 - 34.6 at flow rates calculated to obtain a shear stress of 15 dynes /cm2. At flow rates calculated for a shear stress of 100 dynes/ cm2, the Reynolds number of the chambers ranged from 90.4 - 234. All of these Reynolds numbers are much lower than 2300 and meet the criterion for laminar flow.
Second, for the velocity field and shear stress to be independent of the distance along the flow channel (i.e. fully developed), the distance from the fluid inlet to slide must be longer than the entrance length, Le. This can be satisfied by calculating the entrance length, according to equation 59.
<img alt="Equation 5" src="/files/ftp_upload/3349/3349eq5.jpg" />
For the values listed above, the entrance length ranges from 0.01 to 0.25 cm.
Third, in order to ensure that the velocity and shear stress in the lateral direction do not vary significantly from the value for one-dimensional channel flow (&Delta;Ph/2L), the ratio h/w must be much less than 1. For the average wall shear stress under two-dimensional flow conditions to be 95% of the wall shear stress under one-dimensional flow, h/w must be equal to 0.10, and for the wall shear stress under two-dimensional flow conditions to be 97.5% of the wall shear stress under one-dimensional flow, h/w must be equal to 0.05. With the dimensions of our designed flow chamber 1.7 cm in width and 166 - 267 &mu;m in height, these criteria are satisfied.
The pressure will vary only in the direction of flow if there are no lateral pressure gradients at the entrance. This can be assessed using dyes or particles in the flow path. Further, for steady flow experiments, a pulse dampener is inserted in the flow circuit. The pulse dampener takes out most of the pulsatility caused by the roller pump in the circuit, and allows us to approximate the assumption of steady flow. Of note, the pulse dampener utilized should be compatible with the pump and tubing used in the circuit, so that it can effectively eliminate pulsations in the output flow for the specific frequencies of the roller pump. In our demonstration the Masterflex L/S pulse dampener achieves laminar flow when used on the discharge line with any Masterflex L/S series pump (0 - 600 RPMs) and I/P 26 tubing. For pulsatile flow, a programmable pump can be used to generate various waveforms.
For pulsatile flow, a programmable pump can be used to generate various waveforms.
Furthermore, the circuit is designed such that samples of perfusate can easily be collected at different time points without risking contamination of the cells or flow medium. In our example, the concentration of NO2- was measured by chemiluminescence with an Ionics/Sievers Nitric Oxide Analyzer (NOA 280, Sievers Instruments, Boulder, CO) as previously described10. The reductant used for nitrite analysis was potassium iodide in acetic acid (14.5 M acetic acid and 0.05 M KI), which has the reduction potential to convert nitrite to NO but is insufficient to reduce any higher oxides of nitrogen such as nitrate and thus is relatively specific for nitrite. The total amount nitrite produced was calculated as the product of concentration produced and the total volume of the circuit while adjusting for volume lost while taking samples6.
The following steps are critical for the successful execution of flow experiments:
<ol class="lroman">
	<li>It is desirable to avoid bubble formation during the flow set-up, since bubbles have the potential to tear off cells from their surface. This can be avoided by taking care when placing the top part of the flow chamber onto the bottom part, which is best accomplished by keeping both parallel to each other and lowering the top onto the bottom in one motion without readjustments. In doing so, small air bubbles that may be present and/ or floating in the flow channel, will be diverted into the bubble traps on either end of the aluminum housing.</li>
	<li>It is important to ensure that the outflow tubing in the reservoir reaches down to the reservoir's bottom. Otherwise, air may be sucked into the tubing that leads from reservoir to chamber if the perfusate level falls slightly below the tubing end.</li>
	<li>The researcher should be familiar with the direction of the pump flow so that the pump does not accidentally run in the opposite direction upon commencement of flow, which would result in air being sucked into the circuit and chamber.</li>
	<li>To avoid the formation of foam (due to denatured proteins contained in serum), we advise lowering the tubing that leads from chamber to reservoir to approximately one inch above the perfusate level inside the reservoir. However, keeping it above the flow medium level allows you to verify flow into the reservoir during the experiment.</li>
	<li>When screwing the chamber parts together, it is important to firmly grasp the housing with one hand while operating the electric screwdriver with your other hand. Note that the motion of the electric screwdriver will be translated into torque which has the potential to 'fling the chamber around' once the screw is tightened.</li>
	<li>To avoid any pressure waves from forming in the circuit and lifting cells off their surface, ensure that all stopcocks are open before commencing flow.</li>
	<li>Especially for longer term flow studies (&gt; 48 hours) we recommend using the harder and more resilient tubing to be inserted into the pump head to prevent breaking of tubing.</li>
	<li>It is possible that tubing 'migrates' inside the pump head due to ill-adjusted pump head teeth. Therefore we suggest paying close attention to how the tubing is secured in the pump head and marking each end of the tubing with a marking pen such that you can easily notice if the tubing is 'pulled in' or dislodged during the experiment.</li>
	<li>Always carefully check every single connector of the circuit and chamber prior to starting the perfusion in order to prevent leakage of perfusate during an experiment due to a faulty connection. This is especially important for the inflow and outflow connectors of pulse dampeners!</li>
	<li>Remember to limit light exposure if you use fluorescent labels.</li>
</ol>
A possible limitation of our flow chamber is that the height is fixed by the height of the channel machined into the aluminum. However, this has the advantage of not having to verify and adjust the height of the channel prior to each experiment and therefore simplifies the shear stress calculations by merely adjusting the pump flow to the desired value. Depending on your research goals, it may be desirable to increase the shear stress without increasing pump speed. In this case we recommend increasing the perfusate's viscosity, e.g. adding dextran to the medium11.
A possible limitation of the flow circuit is the large volume of medium used, which can be problematic when attempting to quantify very small concentrations of cell metabolites. Though not shown here, it is possible to substantially reduce the circuit volume by using a smaller reservoir and pulse dampener and decrease tubing length and diameter.
Additionally, there are several other commercially available systems that can be used to apply fluid shear stress to cells in culture. Microfluidic-based systems, e.g. the BioFlux system from Fluxion, enable simultaneous analyses of cells in different microfluidic flow channels loaded with solution into well plates acting as input and output reservoirs for these channels12,13,14. However, these and other microfluidic systems are not compatible with standard microscope slides and do not allow for recovery of a sufficiently large number of cells for further experiments, such as RT-PCR or Western Blot. Further, they are less user-friendly, cost a minimum of $40,000 and may reach a total of more than $100,000, depending on accessory equipment.
Two macrofluidic systems available from the Flexcell International Corporation, the Flexcell Streamer and the FlexFlow systems, have been successfully used to study endothelial cells15,16,17, human annulus cells18 and fibroblasts19 under fluid flow conditions. A third system, available through GlycoTech, has been utilized to study tumor cell adhesion20 and leukocyte adhesion21 to endothelial monolayers.
The Streamer system allows several slides to be run under the same shear stress conditions at once, but lacks a viewing window and - unlike our design - does not allow for real-time visualization of cells under flow.
The FlexFlow system has a viewing window, but requires an upright microscope, which might not be the standard microscope used in most laboratories. Further, the FlexFlow system requires a cell-coated cover slip to be inverted when placed into the flow chamber. This precludes visualization of fluorescent cells on an opaque surface, such as titanium-coated glass, which we demonstrate in our study. Lastly, the specialized cover slips need to be purchased specifically for the FlexFlow system, which is in the multi-thousand-dollar price range, similar to the Flexcell Streamer system.
GlycoTech offers circular and rectangular parallel-plate flow chambers, which are significantly less expensive, but manufactured from cast acrylic that cannot conveniently be stem autoclaved like our chamber. Of note, other flow chambers that have been described to be autoclavable appear impractical because they require special microscopic lenses22,23. The GlycoTech system utilizes silicon rubber gaskets interposed between top and bottom plates, which will change in thickness with repeated use and therefore change chamber height over time (the manufacturer recommends purchasing new ones after every ten uses). Our aluminum chamber with built-in O-rings allows for complete opposition of top and bottom plates and ensures constant chamber height between experiments. Lastly, vacuum pumps are necessary to achieve a leak-proof seal in many flow chamber designs, including the GlycoTech chambers, which are not necessary in our design.
Whereas not shown here, the flow chamber can be kept under a microscope during the entire flow experiment for real-time imaging of cell adhesion and/ or behavior. If this is desired, we recommend using heat lamps or a heated pad under the chamber to maintain the perfusate temperature at 37 &deg;C. Further, the roller pump can be replaced with a syringe pump, if no 'recirculation' of either cells or metabolites or investigational drugs or agents is desired24.
It is also possible to flow differently labeled cells over adherent cells, e.g. fluorescent platelets over a layer of confluent EPCs (using the platelet assay described by Achneck et al.6,25) to evaluate cell-to-cell interaction under fluid shear stress. Our flow chamber combines valuable features of other available flow chambers, such as a perfusate sampling port and a viewing window and has the important advantage of compatibility with either an inverted or upright microscope. It is fully autoclavable and allows for repeated experiments at constant chamber height and without the need of vacuum pumps to achieve a leakproof seal.

<table border="1">
	<tbody>
		<tr>
			<td>Products / Reagents </td>
			<td>Company </td>
			<td>Product #</td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0794012&pfx=EW" target="_blank">10 ml Syringe</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0794012&pfx=EW" target="_blank">07940-12</a></td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=3060000&pfx=EW" target="_blank">1-Way Stopcoks</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=3060000&pfx=EW" target="_blank">30600-00</a></td>
		</tr>
		<tr>
			<td>30 ml Syringe</td>
			<td>BD Medical</td>
			<td>309650</td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=3060004&pfx=EW" target="_blank">4 -Way Stopcocks</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=3060004&pfx=EW" target="_blank">30600-04</a></td>
		</tr>
		<tr>
			<td>Aluminum Alloy</td>
			<td>N/A</td>
			<td>6061</td>
		</tr>
		<tr>
			<td>Cell-Culture Dishes (4-well, rectangular)</td>
			<td>Thermo Scientific; Nunc</td>
			<td>267061</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen</td>
			<td>C34551</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Research Organics</td>
			<td>2162D</td>
		</tr>
		<tr>
			<td>DPBS (-/-)</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
		</tr>
		<tr>
			<td>EGM-2 Singlequots</td>
			<td>Lonza</td>
			<td>CC-4176</td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=4550004&pfx=EW" target="_blank">Female Luer Adaptor</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=4550004&pfx=EW" target="_blank">SI-45500-04</a></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F0895-2MG</td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=3459424&pfx=EW" target="_blank">Glass Bottle (250ml) with Cap</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=3459424&pfx=EW" target="_blank">34594-24</a></td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0650816&pfx=EW" target="_blank">Hard Tubing</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0650816&pfx=EW" target="_blank">06508-16</a></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H8264-500ML</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>H8889-500ML</td>
		</tr>
		<tr>
			<td>Hoechst Stain Solution</td>
			<td>Sigma</td>
			<td>H6024</td>
		</tr>
		<tr>
			<td>Hyclone FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30071.01</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Lonza</td>
			<td>17-605E</td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=4550504&pfx=EW" target="_blank">Male Luer Adaptor</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=4550504&pfx=EW" target="_blank">EW-45505-04</a></td>
		</tr>
		<tr>
			<td>MCDB-131, 1X Medium</td>
			<td>Cellgro</td>
			<td>15-100-CV</td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0636590&pfx=EW" target="_blank">Barbed Polypropylene Fittings</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0636590&pfx=EW" target="_blank">06365-90</a></td>
		</tr>
		<tr>
			<td>O-Rings</td>
			<td>N/A</td>
			<td>AS568A</td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0759620&pfx=EW" target="_blank">Pulse-Dampener</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0759620&pfx=EW" target="_blank">07596-20</a></td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0752440&pfx=EW" target="_blank">Pump-Drive (Masterflex L/S variable-speed economy drive)</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0752440&pfx=EW" target="_blank">7524-40</a></td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0751860&pfx=EW" target="_blank">Pump-Head (Masterflex Easy Load Pump Head)</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0751860&pfx=EW" target="_blank">07518-60</a></td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=4850000&pfx=EW" target="_blank">Slide</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=4850000&pfx=EW" target="_blank">48500-00</a></td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=9640016&pfx=EW" target="_blank">Soft Tubing</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=9640016&pfx=EW" target="_blank">96400-16</a></td>
		</tr>
		<tr>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0291512&pfx=EW" target="_blank">Syringe filter</a></td>
			<td>Cole Parmer Instrument Co.</td>
			<td><a href="http://www.coleparmer.com/catalog/product_view.asp?sku=0291512&pfx=EW" target="_blank">02915-12</a></td>
		</tr>
		<tr>
			<td>Sytox Orange Nucleic Acid Stain</td>
			<td>Invitrogen</td>
			<td>S-11368</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Lonza</td>
			<td>CC-5012</td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
		</tr>
	</tbody>
</table>

parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

Watch this Scientific Journal Video about Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress at JoVE.com

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

parallel-plate-flow-chamber-continuous-flow-circuit-to-evaluate

Research

JoVE Journal

Bioengineering

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

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 ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...