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. Cellodling och Förbehandling <ol><li> Skära 3-tums cirkulära substrat från en 100 x 20 mm vävnadsodlingsskål (BD Falcon) med användning av en svarv. Sterilisera substrat genom nedsänkning i 70% etanol. Rum i en Petri-skål och belägga med 4 ml typ-I-kollagen (100 | ig / ml) under 1 h vid rumstemperatur och sköljs sedan med 4 ml 1 x PBS. </li><li> Suspendera humana aortaendotelceller (HAEC, passagen 4-6) vid 6.5x10 5 celler per ml och frö genom applicering 1 ml direkt på substratet. Rum i en 3...

<ol>
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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

Aterogenes potentieras av metabola störningar som bidrar till en ökad tillstånd av systemisk inflammation resulterar i endotel dysfunktion. Dock tidiga funktionella förändringar i endotelet som betyder en individs risknivå inte direkt bedömas kliniskt för att vägleda terapeutisk strategi. Dessutom bidrar regleringen av inflammation av lokala hemodynamiken till den icke-slumpmässiga geografiska fördelningen av åderförkalkning, men mekanismerna är svåra att avgränsa in vivo. Vi beskriver ett lab-on-a-chip metod för att kvantitativt analys metabolisk störning av inflammatoriska händelser i humana endotelceller (EG) och monocyter enligt exakta flöde. Standardmetoder för mjuk litografi används för att microfabricate vaskulära mimetiska mikrofluidiska kammare (VMMC), som är bundna direkt till odlade EC monoskikt. 1 Dessa anordningar har fördelen med att använda små volymer av reagens medantillhandahålla en plattform för direkt avbildning av inflammatoriska händelser på membranet i EG utsätts för en väldefinierad skjuvning området. Vi har framgångsrikt tillämpat dessa enheter för att undersöka cytokin-, 2 lipid-3, 4 och RAGE-inducerad 5 inflammation i mänsklig aorta EG (HAEC). Här har vi dokumentera användningen av VMMC att analysera monocytisk cell (THP-1) valsning och gripande om HAEC monolager som konditioneras under egenskaper differentiella skjuv-och aktiveras av inflammatoriska cytokin TNF-α. Studier som dessa tillhandahåller mekanistiska inblick i atherosusceptibility i metabola riskfaktorer.

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.

On-Chip Endothelial inflammatorisk Fenotypning

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

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