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

Questo lavoro è stato supportato dal NIH / NHLBI concessione R01 HL082689 a Scott I. Simon e Anthony G. Passerini.

1. Di coltura cellulare e Preparazione del substrato <ol><li> Tagliare supporti circolari da 3 pollici da una x 100 20 piatto di coltura tissutale mm (BD Falcon) usando un tornio. Sterilizzare substrati mediante immersione in etanolo al 70%. Posto in una capsula di Petri e mantello con 4 ml di collagene di tipo I (100 pg / ml) per 1 ora a temperatura ambiente, poi lavare con 4 ml 1 x PBS. </li><li> Sospendere Le cellule endoteliali aortiche (HAEC, passaggio 4-6) a 6.5x10 5 cellule / ml e di sementi mediante...

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	<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. P., Schaff, U. Y., Hui, D. Y., Rutledge, J. C., Knowlton, A. A., 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=Triglyceride-rich+lipoproteins+prime+aortic+endothelium+for+an+enhanced+inflammatory+response+to+tumor+necrosis+factor-alpha.">Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.</a> Circ. Res. 100, 381-390 (2007).</li><li>Wang, Y. I., Schulze, J., Raymond, N., Tomita, T., Tam, K., Simon, S. I., 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=Endothelial+inflammation+correlates+with+subject+triglycerides+and+waist+size+after+a+high-fat+meal.">Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal.</a> Am. J. Physiol. Heart Circ. 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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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+heterogeneity+associated+with+regional+athero-susceptibility+and+adaptation+to+disturbed+blood+flow+in+vivo.">Endothelial heterogeneity associated with regional athero-susceptibility and adaptation to disturbed blood flow in vivo.</a> Semin Thromb Hemost. 36, 265-275 (2008).</li><li>Burdge, G. C., Calder, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cytokine+response+during+the+postprandial+period:+A+potential+causal+process+in+vascular+disease.">Plasma cytokine response during the postprandial period: A potential causal process in vascular disease.</a> Br. J. Nutr. 93, 3-9 (2005).</li><li>Hotamisligil, G. 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. 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Si descrivono l&#39;uso di dispositivi microfluidici PDMS per la valutazione on-chip di endoteliale fenotipo infiammatoria attraverso l&#39;imaging in tempo reale di espressione CAM e adesione monociti. Uno dei principali vantaggi del nostro approccio risiede nella capacità di quantificare i risultati associati a disfunzione endoteliale in cellule esposte a mediatori infiammatori come i lipidi alimentari e citochine in condizioni idrodinamiche definiti che rispecchiano shear stress nei vasi aterogene. Il VMMC facilitar...

<table border="1"><tbody><tr><td> Voce </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> 100 x 20mm piastre di Petri </td><td> BD Falcon </td><td> 353003 </td></tr><tr><td> Etanolo 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> Collagene di tipo I Tail derivata Rat </td><td> Gibco </td><td> A10483-01 </td></tr><tr><td> Cellule endoteliali umane aortica </td><td> Genlantis </td><td> PH30405A </td></tr><tr><td> Antibiotico-antimicotica Solution </td><td> Invitrogen </td><td> 15240-062 </td></tr><tr><td> BulletKit endoteliale </td><td> Lonza </td><td> CC-4176 </td></tr><tr><td> Endoteliali basali Media-2 </td><td> Lonza </td><td> CC-3156 </td></tr><tr><td> Siringhe da 10 ml </td><td> BD Falcon </td><td> 309604 </td></tr><tr><td> Tubi in poliuretano </td><td> Tygon </td><td> ABW0001 </td></tr><tr><td> Leibovitz-15 supporti </td><td> Gibco </td><td> 11415-069 </td></tr><tr><td> Sylgard 184 Base in silicone elastomero </td><td> Dow Corning </td><td> 184 </td></tr><tr><td> Sylgard 184 elastomero siliconico Catalizzatore </td><td> Dow Corning </td><td> 184 </td></tr><tr><td> SU8 Photoresist Maestro Wafer </td><td> UC Davis Pan Lab </td><td> N / A </td></tr><tr><td> Eclipse TE200 microscopio invertito </td><td> Nikon </td><td> Eclipse TE200 </td></tr><tr><td> Pompa a siringa </td><td> Harvard Apparatus </td><td> PHD2000 </td></tr><tr><td> 19 gauge ago ipodermico </td><td> Kendall </td><td> 8881 </td></tr><tr><td> THP-1 linea cellulare monocitica </td><td> ATCC </td><td> TIB-202 </td></tr><tr><td> HBSS (Hanks soluzione salina tamponata) con Ca 2 + / Mg 2 + </td><td&gt; Gibco </td><td> 14025-092 </td></tr><tr><td> Fattore di Necrosi Tumorale alfa (TNF-α) </td><td> R &amp; D Systems </td><td> 210-TA-010 </td></tr><tr><td> Stromal Factor Derivato - 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> Albumina serica umana (HSA) </td><td> ZLB Behring </td><td> NDC 0053-7680-32 </td></tr></tbody></table> Tabella 2. Reagenti e attrezzature specifiche.

on-chip endothelial inflammatory phenotyping

Aterogenesi è potenziato da alterazioni metaboliche che contribuiscono ad un elevato stato di infiammazione sistemica con conseguente disfunzione endoteliale. Tuttavia, i primi cambiamenti funzionali dell&#39;endotelio che indicano il livello individuale di rischio non sono direttamente valutato clinicamente per aiutare strategia terapeutica guida. Inoltre, la regolazione di infiammazione emodinamica locali contribuisce alla non casuale distribuzione spaziale di aterosclerosi, ma i meccanismi sono difficili da delineare in vivo. Descriviamo un approccio lab-on-a-chip basato su test di quantitativamente perturbazione metabolica di eventi infiammatori nelle cellule endoteliali umane (CE) e dei monociti in condizioni di flusso precise. Metodi standard di litografia soft sono usati per microfabricate vascolari camere mimetici microfluidica (VMMC), che sono legati direttamente alla coltura monostrati CE. 1 Questi dispositivi hanno il vantaggio di utilizzare piccoli volumi di reagenti, mentrefornendo una piattaforma per l&#39;imaging direttamente gli eventi infiammatori a livello della membrana di CE esposto ad un ben definito campo di taglio. Abbiamo applicato con successo questi dispositivi per indagare citochine, lipidi 2-3, 4 e 5 RAGE indotta infiammazione in umano aortica CE (HAEC). Qui documentare l&#39;uso della cella VMMC di dosaggio monocitiche (THP-1) laminazione e arresto su monostrati HAEC che sono condizionati alle caratteristiche di taglio differenziali e attivato dalla citochina infiammatoria TNF-α. Studi come questi stanno fornendo informazioni meccanicistica in atherosusceptibility in caso di fattori di rischio metabolici.

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

On-Chip Endothelial Inflammatory Phenotyping

Camere di flusso Microfluidic incisi da fotolitografia e fabbricati da PDMS sono applicate per sondare gli esiti funzionali associati alla disfunzione CE e l&#39;infiammazione. In un esperimento rappresentativo, la capacità di sforzo di taglio differenziale di modulare l&#39;adesione cellulare monocitica di citochina attivato CE monostrati è dimostrata.

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

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