Name: on-chip endothelial inflammatory phenotyping
Uploaded: 2012-07-21T18:00:00
Description: Watch this Scientific Journal Video about On-Chip Endothelial Inflammatory Phenotyping at JoVE.com

This work was supported by NIH/NHLBI grant R01 HL082689 to Scott I. Simon and Anthony G. Passerini.

1. Cell Culture and Substrate Preparation
 <ol>
 <li>Cut 3-inch circular substrates from a 100 x 20 mm tissue culture dish (BD Falcon) using a lathe. Sterilize substrates by submersion in 70% ethanol. Place in a Petri dish and coat with 4 ml type-I collagen (100 &mu;g/ml) for 1 hr at room temperature, then rinse with 4 ml 1 x PBS. </li>
 <li>Suspend Human Aortic Endothelial Cells (HAEC, passage 4-6) at 6.5x105 cells/ml and seed by applying 1 ml directly to substrate. Place in a 37 &deg;C, 5% CO<s...

<ol>
	<li>Schaff, U. Y., Xing, M. M., Lin, K. K., Pan, N., Jeon, N. L., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+mimetics+based+on+microfluidics+for+imaging+the+leukocyte--endothelial+inflammatory+response.">Vascular mimetics based on microfluidics for imaging the leukocyte--endothelial inflammatory response.</a> Lab Chip. 7, 448-456 (2007).</li><li>Tsou, J. K., Gower, R. M., Ting, H. J., Schaff, U. Y., Insana, M. F., Passerini, A. G., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+inflammation+by+human+aortic+endothelial+cells+in+a+linear+gradient+of+shear+stress.">Spatial regulation of inflammation by human aortic endothelial cells in a linear gradient of shear stress.</a> Microcirculation. 15, 311-323 (2008).</li><li>Ting, H. J., Stice, J. 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Physiol. 300, H784-H791 (2011).</li><li>Deverse, J. S., Bailey, K. A., Jackson, K. N., Passerini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+modulates+rage-mediated+inflammation+in+a+model+of+diabetes-induced+metabolic+stress.">Shear stress modulates rage-mediated inflammation in a model of diabetes-induced metabolic stress.</a> Am. J. Physiol. Heart Circ. Physiol. , (2012).</li><li>Dai, G., Kaazempur-Mofrad, M. R., Natarajan, S., Zhang, Y., Vaughn, S., Blackman, B. R., Kamm, R. D., Garcia-Cardena, G., Gimbrone, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+metabolic+disorders.">Inflammation and metabolic disorders.</a> Nature. 444, 860-867 (2006).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+atherosclerosis.">Inflammation in atherosclerosis.</a> Nature. 420, 868-874 (2002).</li><li>Nigro, P., Abe, J., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+shear+stress+and+atherosclerosis:+A+matter+of+site+specificity.">Flow shear stress and atherosclerosis: A matter of site specificity.</a> Antioxid Redox Signal. 15, 1405-1414 (2011).</li><li>Chiu, J. J., Lee, P. L., Chen, C. N., Lee, C. I., Chang, S. F., Chen, L. J., Lien, S. C., Ko, Y. C., Usami, S., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+increases+icam-1+and+decreases+vcam-1+and+e-selectin+expressions+induced+by+tumor+necrosis+factor-[alpha]+in+endothelial+cells.">Shear stress increases icam-1 and decreases vcam-1 and e-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells.</a> Arterioscler. Thromb. Vasc. Biol. 24, 73-79 (2004).</li><li>Li, Y. S., Haga, J. H., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+effects+of+shear+stress+on+vascular+endothelial+cells.">Molecular basis of the effects of shear stress on vascular endothelial cells.</a> J. Biomech. 38, 1949-1971 (2005).</li><li>Usami, S., Chen, H. H., Zhao, Y., Chien, S., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+construction+of+a+linear+shear+stress+flow+chamber.">Design and construction of a linear shear stress flow chamber.</a> Ann. Biomed. Eng. 21, 77-83 (1993).</li><li>Helmke, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+control+of+cytoskeletal+mechanics+by+hemodynamic+forces.">Molecular control of cytoskeletal mechanics by hemodynamic forces.</a> Physiology (Bethesda). 20, 43-53 (2005).</li><li>Gower, R. M., Wu, H., Foster, G. A., Devaraj, S., Jialal, I., Ballantyne, C. M., Knowlton, A. A., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cd11c/cd18+expression+is+upregulated+on+blood+monocytes+during+hypertriglyceridemia+and+enhances+adhesion+to+vascular+cell+adhesion+molecule-1.">Cd11c/cd18 expression is upregulated on blood monocytes during hypertriglyceridemia and enhances adhesion to vascular cell adhesion molecule-1.</a> Arterioscler. Thromb. Vasc. Biol. 31, 160-166 (2011).</li><li>Wu, H., Gower, R. M., Wang, H., Perrard, X. Y., Ma, R., Bullard, D. C., Burns, A. R., Paul, A., Smith, C. W., Simon, S. I., Ballantyne, C. 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We describe the use of microfluidic PDMS devices for the on-chip assessment of endothelial inflammatory phenotype through the real-time imaging of CAM expression and monocyte adhesion. A major advantage of our approach lies in the ability to quantify outcomes associated with endothelial dysfunction in cells exposed to inflammatory mediators such as dietary lipids and cytokines under defined hydrodynamic conditions that mirror shear stress in atherogenic vessels. The VMMC facilitate the use of small amounts of reagents or...

<table border="1">
<tbody>
 <tr>
 <td>Item</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>100 x 20mm Petri Dishes</td>
 <td>BD Falcon</td>
 <td>353003</td>
 </tr>
 <tr>
 <td>Ethanol 95%</td>
 <td>EMD Chemicals</td>
 <td>EX0290-1</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Cellgro</td>
 <td>21-031-CV</td>
 </tr>
 <tr>
 <td>Type I Rat Tail Derived Collagen</td>
 <td>Gibco</td>
 <td>A10483-01</td>
 </tr>
 <tr>
 <td>Human Aortic Endothelial Cells</td>
 <td>Genlantis</td>
 <td>PH30405A</td>
 </tr>
 <tr>
 <td>Antibiotic-Antimycotic Solution</td>
 <td>Invitrogen</td>
 <td>15240-062</td>
 </tr>
 <tr>
 <td>Endothelial BulletKit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 </tr>
 <tr>
 <td>Endothelial Basal Media-2</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 </tr>
 <tr>
 <td>10 ml Syringes</td>
 <td>BD Falcon</td>
 <td>309604 </td>
 </tr>
 <tr>
 <td>Polyurethane tubing</td>
 <td>Tygon</td>
 <td>ABW0001</td>
 </tr>
 <tr>
 <td>Leibovitz-15 Media</td>
 <td>Gibco</td>
 <td>11415-069</td>
 </tr>
 <tr>
 <td>Sylgard 184 Silicone Elastomer Base</td>
 <td>Dow Corning</td>
 <td>184</td>
 </tr>
 <tr>
 <td>Sylgard 184 Silicone Elastomer Curing Agent</td>
 <td>Dow Corning</td>
 <td>184</td>
 </tr>
 <tr>
 <td>SU8 Photoresist Master Wafer</td>
 <td>UC Davis Pan Lab</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>Eclipse TE200 Inverted Microscope</td>
 <td>Nikon</td>
 <td>Eclipse TE200</td>
 </tr>
 <tr>
 <td>Syringe Pump</td>
 <td>Harvard Apparatus</td>
 <td>PHD2000</td>
 </tr>
 <tr>
 <td>19 gauge hypodermic needle</td>
 <td>Kendall</td>
 <td>8881</td>
 </tr>
 <tr>
 <td>THP-1 Monocytic Cell Line</td>
 <td>ATCC</td>
 <td>TIB-202</td>
 </tr>
 <tr>
 <td>HBSS (Hanks Buffered Saline Solution) with Ca2+/Mg2+ </td>
 <td>Gibco</td>
 <td>14025-092</td>
 </tr>
 <tr>
 <td>Tumor Necrosis Factor Alpha (TNF-&alpha;)</td>
 <td>R&amp;D Systems</td>
 <td>210-TA-010</td>
 </tr>
 <tr>
 <td>Stromal Derived Factor - 1 (SDF-1)</td>
 <td>R&amp;D Systems</td>
 <td>350-NS-010</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Cellgro </td>
 <td>10-040-CV</td>
 </tr>
 <tr>
 <td>Human Serum Albumin (HSA)</td>
 <td>ZLB Behring</td>
 <td>NDC 0053-7680-32</td>
 </tr>
 </tbody>
</table>
Table 2. Specific reagents and equipment.

on-chip endothelial inflammatory phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Watch this Scientific Journal Video about On-Chip Endothelial Inflammatory Phenotyping at JoVE.com

On-Chip Endothelial Inflammatory Phenotyping

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

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