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

Dit werk werd ondersteund door NIH / NHLBI subsidie ​​R01 HL082689 van Scott I. Simon en Anthony G. Passerini.

1. Cel Cultuur en Voorbereiding van de ondergrond <ol><li> Snijd 3-inch ronde substraten van een 100 x 20 mm weefselkweek schotel (BD Falcon) met behulp van een draaibank. Steriliseer substraten door onderdompeling in 70% ethanol. Plaats in een petrischaal en mantel met 4 ml type I collageen (100 ug / ml) gedurende 1 uur bij kamertemperatuur spoel met 4 ml 1 x PBS. </li><li> Hang Human Aorta endotheelcellen (HAEC, passage 4-6) op 6.5x10 5 cellen / ml en het zaad door het toepassen van 1 ml direct op substra...

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We beschrijven het gebruik van microfluïdische PDMS apparaten voor de on-chip beoordeling van de endotheliale inflammatoire fenotype door de real-time beeldvorming van CAM expressie en monocyten hechting. Een groot voordeel van onze aanpak ligt in het vermogen om de resultaten in verband met disfunctioneren van het endotheel in de cellen blootgesteld aan ontstekingsmediatoren zoals dieet lipiden en cytokines onder gedefinieerde hydrodynamische omstandigheden die shear stress een afspiegeling van de atherogene schepen t...

<table border="1"><tbody><tr><td> Item </td><td> Vennootschap </td><td> Catalogus nummer </td></tr><tr><td> 100 x 20 mm Petrischalen </td><td> BD Falcon </td><td> 353003 </td></tr><tr><td> Ethanol 95% </td><td> Merck Chemicals </td><td> EX0290-1 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21 tot 031-CV </td></tr><tr><td> Type I Rat Tail Afgeleide Collageen </td><td> Gibco </td><td> A10483-01 </td></tr><tr><td> Human Aorta endotheelcellen </td><td> Genlantis </td><td> PH30405A </td></tr><tr><td> Antibiotica-Antimycoticum Solution </td><td> Invitrogen </td><td> 15240-062 </td></tr><tr><td> Endotheliale BulletKit </td><td> Lonza </td><td> CC-4176 </td></tr><tr><td> Endotheliale Basale Media-2 </td><td> Lonza </td><td> CC-3156 </td></tr><tr><td> 10 ml spuiten </td><td> BD Falcon </td><td> 309604 </td></tr><tr><td> Polyurethaan slang </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-elastomeer Base </td><td> Dow Corning </td><td> 184 </td></tr><tr><td> Sylgard 184 siliconelastomeerfase Verharder </td><td> Dow Corning </td><td> 184 </td></tr><tr><td> SU8 fotolak Master Wafer </td><td> UC Davis Pan Lab </td><td> N / A </td></tr><tr><td> Eclipse TE200 omgekeerde microscoop </td><td> Nikon </td><td> Eclipse TE200 </td></tr><tr><td> Spuitpomp </td><td> Harvard Apparatuur </td><td> PHD2000 </td></tr><tr><td> 19 gauge injectienaald </td><td> Kendall </td><td> 8881 </td></tr><tr><td> THP-1 monocytische cellijn </td><td> ATCC </td><td> TIB-202 </td></tr><tr><td> HBSS (Hanks zoutoplossing) met Ca2 + / Mg2 + </td><td&gt; Gibco </td><td> 14025-092 </td></tr><tr><td> Tumor Necrosis Factor Alfa (TNF-α) </td><td> R &amp; D Systems </td><td> 210-TA-010 </td></tr><tr><td> Stromale afgeleide 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 tot 040-CV </td></tr><tr><td> Humaan serum albumine (HSA) </td><td> ZLB Behring </td><td> NDC 0053-7680-32 </td></tr></tbody></table> Tabel 2. Specifieke reagentia en materialen.

on-chip endothelial inflammatory phenotyping

Atherogenese wordt versterkt door metabole afwijkingen die bijdragen aan een verhoogde staat van systemische ontsteking leidt tot disfunctioneren van het endotheel. Echter, begin van de functionele veranderingen in endotheel, dat betekent een individu niveau van het risico niet direct klinisch onderzocht naar de gids van therapeutische strategie te helpen. Bovendien, de regulering van ontsteking door lokale hemodynamica draagt ​​de niet-random ruimtelijke verdeling van atherosclerose, maar de mechanismen moeilijk af te bakenen in vivo. We beschrijven een lab-on-a-chip gebaseerde benadering van kwantitatief onderzoek metabole verstoring van inflammatoire gebeurtenissen in de menselijke endotheelcellen (EC) en monocyten in een zeer nauwkeurige voorwaarden. Standaardmethoden van zachte lithografie worden vasculaire mimetische microfluïdische kamers (VMMC), die direct gebonden aan gekweekte EC monolagen microfabricate. Een deze inrichtingen hebben het voordeel van het gebruik van kleine hoeveelheden reagentia terwijleen platform voor direct beeld van de ontstekingen in het membraan van EC blootgesteld aan een welbepaalde shear veld. We hebben succes toegepast deze apparaten cytokine, 2 lipide-3, 4 en RAGE-geïnduceerde 5 ontsteking in menselijke aorta EC (HAEC) onderzoeken. Hier aantonen hoe de VMMC voor het testen van monocyten cel (THP-1) wals en stilstand van HAEC monolagen die geconditioneerd onder differentiële afschuiving eigenschappen en geactiveerd door inflammatoire cytokine TNF-α. Studies zoals deze zijn het verstrekken van mechanistisch inzicht in atherosusceptibility onder metabolische risicofactoren.

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

On-Chip Endothelial Inflammatory Phenotyping

Microfluïdische stromingskamers geëtst door fotolithografie en vervaardigd uit PDMS worden toegepast op functionele resultaten in verband met EG-dysfunctie en ontsteking te onderzoeken. In een representatief experiment het vermogen van differentiële schuifspanning moduleren monocyte celhechting aan cytokine geactiveerd EC monolagen wordt aangetoond.

On-Chip Endotheliale Inflammatoire Fenotypering

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

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