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 ...

Research

JoVE Journal

Bioengineering

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