Name: the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro
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Forfatterne takke de følgende medarbejdere og forskning personale: Andrée Gauthier-Fisher, Matthew Librach, Tanya Barretto, Tharsan Velauthapillai og Sarah Laronde.

alle undersøgelser, der involverer dyr blev gennemført og rapporteret efter ANKOMME retningslinjer 29. Alle undersøgelser blev udført med institutionelle forskning etik bestyrelsen godkendelse (REB antallet 4276). Alle dyr procedurer blev godkendt af Animal Care Committee of University Health Network (Toronto, Canada), og alle dyr modtages Human pleje i overensstemmelse med den vejledning for pleje og brugen af forsøgsdyr, 8 th edition ( National institutes of Health 201...

<ol>
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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+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90 (3), 195-221 (2009).</li><li>Auerbach, R., Lewis, R., Shinners, B., Kubai, L., Akhtar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+assays:+a+critical+overview.">Angiogenesis assays: a critical overview.</a> Clin Chem. 49 (1), 32-40 (2003).</li><li>Tahergorabi, Z., Khazaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+angiogenesis+and+its+assays.">A review on angiogenesis and its assays.</a> Iran J Basic Med Sci. 15 (6), 1110-1126 (2012).</li><li>Arnaoutova, I., George, J., Kleinman, H. K., Benton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+cell+tube+formation+assay+on+basement+membrane+turns+20:+state+of+the+science+and+the+art.">The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art.</a> Angiogenesis. 12 (3), 267-274 (2009).</li><li>Arnaoutova, I., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nat Protoc. 5 (4), 628-635 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10 (3), 588-612 (2006).</li><li>Nicosia, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aortic+ring+model+of+angiogenesis:+a+quarter+century+of+search+and+discovery.">The aortic ring model of angiogenesis: a quarter century of search and discovery.</a> J Cell Mol Med. 13 (10), 4113-4136 (2009).</li><li>Baker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+mouse+aortic+ring+assay+to+study+angiogenesis.">Use of the mouse aortic ring assay to study angiogenesis.</a> Nat Protoc. 7 (1), 89-104 (2011).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+protein+(Canopy+2,+CNPY2)+enhances+angiogenesis+and+promotes+smooth+muscle+cell+migration+and+proliferation.">A secreted protein (Canopy 2, CNPY2) enhances angiogenesis and promotes smooth muscle cell migration and proliferation.</a> Cardiovasc Res. 105 (3), 383-393 (2015).</li><li>Wittig, C., Scheuer, C., Parakenings, J., Menger, M. D., Laschke, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geraniol+Suppresses+Angiogenesis+by+Downregulating+Vascular+Endothelial+Growth+Factor+(VEGF)/VEGFR-2+Signaling.">Geraniol Suppresses Angiogenesis by Downregulating Vascular Endothelial Growth Factor (VEGF)/VEGFR-2 Signaling.</a> PLoS One. 10 (7), e0131946 (2015).</li><li>Masson, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Aortic+Ring+Assay:+A+New+Approach+of+the+Molecular+Genetics+of+Angiogenesis.">Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis.</a> Biol Proced Online. 4, 24-31 (2002).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells+for+tissue+engineering+versus+regenerative+medicine.">Adult mesenchymal stem cells for tissue engineering versus regenerative medicine.</a> J Cell Physiol. 213 (2), 341-347 (2007).</li><li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+trophic+mediators.">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li><li>Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+new+directions.">Mesenchymal stromal cells: new directions.</a> Cell Stem Cell. 10 (6), 709-716 (2012).</li><li>Kilroy, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+human+adipose-derived+stem+cells:+expression+of+angiogenic,+hematopoietic,+and+pro-inflammatory+factors.">Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors.</a> J Cell Physiol. 212 (3), 702-709 (2007).</li><li>Tang, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+action+enhances+the+effects+of+autologous+mesenchymal+stem+cell+transplantation+on+vascular+regeneration+in+rat+model+of+myocardial+infarction.">Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction.</a> Ann Thorac Surg. 80 (1), 229-236 (2005).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+MSCs+are+pericytes?.">All MSCs are pericytes?.</a> Cell Stem Cell. 3 (3), 229-230 (2008).</li><li>Hong, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+human+umbilical+cord+perivascular+cells:+molecular+and+fate+potential+changes+during+gestation.">Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation.</a> Stem Cells Dev. 22 (17), 2425-2439 (2013).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+The+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research.</a> J Pharmacol Pharmacother. 1 (2), 94-99 (2010).</li><li>Sarugaser, R., Ennis, J., Stanford, W. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+propagation,+and+characterization+of+human+umbilical+cord+perivascular+cells+(HUCPVCs).">Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs).</a> Methods Mol Biol. 482, 269-279 (2009).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Gelati, M., Aplin, A. C., Fogel, E., Smith, K. D., Nicosia, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+angiogenic+response+of+the+aorta+to+injury+and+inflammatory+cytokines+requires+macrophages.">The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages.</a> J Immunol. 181 (8), 5711-5719 (2008).</li></ol>

Der er flere kritiske faser i oprettelse af en vellykket aorta ring assay MSC Co kultur eksperiment. Første, de mest vigtige skridt når isolere og skæring aorta er: 1) at opnå udelukkende thorax segmentet af aorta; 2) omhyggeligt at fjerne forgrening blodkar, bindevæv og fedtvæv og; 3) skære selv dele af aorta (~ 1 mm) til at begrænse variabiliteten mellem hvert assay. Andet, vellykket integrering af aorta ringe på BME er kritisk for denne analyse. Hvis BME er ikke helt polymeriserede eller polymeriserede ujævn...

Den komplekse proces af angiogenese forbedrer og vedligeholder væv perfusion af nyt blod fartøj udvikling fra allerede eksisterende Vaskulaturen1. Det er en stramt reguleret, afbalanceret proces af pro-angiogene og anti-angiogene faktorer. Enhver mangel i dette system kan føre til utilstrækkelig fartøj vedligeholdelse eller vækst, forårsager alvorlige iskæmiske sygdomme herunder Myokardie sygdom, slagtilfælde og neurodegenerative lidelser. Overdrevet vaskulære udvikling er imidlertid karakteristisk for betingelser, herunder kræft og inflammatoriske lidelser2.
Udvikling af behandlinger, der har til formål at kontrollere angiogenese for at opnå gunstige vævsregeneration er af afgørende betydning. Trods omfattende prækliniske og kliniske undersøgelser, har forsøg på at stimulere angiogenese ved hjælp af pro-angiogene faktorer og MicroRNA undladt at opnå ønskede resultater3,4,5. Mulige årsager til forbigående virkninger omfatter: begrænsede levetiden af angiogene proteiner og nukleinsyrer og det begrænsede antal målrettet vækstfaktorer6,7. Selvom opløselige angiogene faktorer er afgørende for at indlede angiogenese, afhænger vedligeholdelse og funktionalitet af Vaskulaturen støtte celletyper, herunder pericytes og glat muskel celler8. Feltet af pro-angiogene behandlingsformer nu udforske potentielle stamceller og stamceller celle kilder, der kan give angiogene faktorer lokalt, mens fysisk støtte nyligt udviklet Vaskulaturen, selvstændig fornyelse eller endda differentiere i endotel-lignende celler9,10. At finde den optimale angiogene celletyper med kapacitet til at opfylde disse funktionelle krav holder en stor lovende for iskæmisk væv revitalisering.
For at med held oversætte potentielle cellebaserede behandlinger i kliniske forsøg, skal prækliniske undersøgelser vise deres effektivitet og fremhæve de underliggende angiogene mekanismer. Trods det høje antal etablerede angiogenese assays, feltet mangler en "guldstandard" i vitro assay, der pålideligt kunne evaluere effekten af potentiel kandidat celle typer11,12, 13. de fleste in vitro- angiogenese assays (herunder i endothelial spredning, migration og tube dannelse assays) typisk vurdere virkningerne af celler eller forbindelser i endothelial celler phenotypical ændringer eller differentiering af rørformede og netværk strukturer14,15. Mens disse funktioner er afgørende for angiogenese, en "oversættes" assay bør også evaluere: 1) brystforstørrelse eller udskiftning af de understøttende celletyper, herunder pericytes eller glatte muskelceller, 2) forarbejdningen af ECM og/eller basalmembranen, og 3). effektivitet til at fremme dannelsen af funktionelle microvasculature. In vivo angiogenese modeller, herunder hornhinde assay og Matrigel plug assay, sammenfatte unikke i vivo mikromiljø men bliver udfordret ved sværhedsgrad af tracking administreres celler til at observere fysiske interaktioner. Desuden, i vivo modeller, xeno-immune afvisning kan forekomme under test potentielle allogene celle terapi kandidater16. Ex vivo angiogenese modeller, især i aorta ring assay kan give: 1) let observation og kvantificering af rørformede strukturer, 2) tilbehør støttende celler, 3) ECM fra vært og kunstige forsyninger, 4) udelukkelsen af inflammatoriske komponenter, og 5) hurtig og billig installation17,18. Typisk, aorta ring assay kan teste lille sekretoriske proteiner og farmakologiske agenser og genetisk modificerede gnavere modeller19,20,21angiogene muligheder.
MSC'er er lovende kandidater til vaskulær regenerering primært gennem deres paracrine-medierede effekter22,23,24. MSC'er har vist sig at udskille centrale angiogene faktorer herunder vaskulær endotel vækstfaktor (VEGF), hepatocyt vækstfaktor (HGF), Insulin-lignende vækstfaktor-1 (IGF-1), basic Fibroblast vækstfaktor (bFGF) og angiopoeitin-1 (Ang-1)25 ,26. MSC'er kan også opdage og hjemsted for iskæmisk eller betændt væv, men de præcise mekanismer er stadig under efterforskning. I stigende grad støtter litteraturen den hypotese, at de fleste MSCs opstår fra perivascular celler, co express pericyte markører og kan opføre sig som pericytes27. HUCPVCs er en ung kilde til MSCs stammer fra perivascular-regionen i den menneskelige navlestrengen. De repræsenterer en befolkning på MSCs med pericyte-lignende egenskaber og har været præget af både FTM og sigt umbilical snore. FTM HUCPVCs viser et højt udtryk af pericyte markører herunder CD146 og NG2, høj proliferativ og multilineage potentiale, immun-privilegeret egenskaber, og vise en robust paracrine profil28. FTM HUCPVCs er en ideel kandidat celletype skal fremme regenerering af tilskadekomne væv gennem fremme af nye Vaskulaturen via deres pericyte-lignende egenskaber.
For at teste angiogene potentiale og pericyte-lignende egenskaber af menneskelige MSC'er, er et meget begrænset antal angiogenese assays tilgængelige hvor positive angiotropic migration (i det følgende benævnt "homing"), ECM forarbejdning og udvikling af fysisk interaktioner mellem celletyper kan undersøges under hentning kvantitative data om microvasculature udvikling.
Vi præsenterer hermed en protokol, der beskriver en roman anvendelse af aorta ring assay. Menneskelige MSCs var Co kulturperler med udviklingslandene rotte-afledte aorta endotel netværk til at vurdere deres bidrag til tube dannelse, modning og homøostase. Denne version af aorta ring analysen vurderer evnen og styrken af celle terapi kandidater til hjem til websteder af angiogenese, udføre og mægle ECM behandling, og bidrage til endotel rørformede udvikling gennem oprettelse af pericyte-lignende fysiske interaktioner. Ud over at kvantificere nettoeffekten af MSCs på in vitro- endotel netværk dannelse og observere intercellulære interaktioner, optimeret vi også en protokol for at isolere MSCs fra Co kulturer. Ved at udføre flowcytometri og qPCR, er det muligt at karakterisere ændringer i MSC fænotype og gen udtryk efter fælles kultur. Som model celletyper, vi sammenlignet ontogenetically tidligt (prænatal) og slutningen (voksen) kilder til menneskelige MSCs: FTM HUCPVCs og menneskelige knoglemarv-afledte MSCs (BMSC), henholdsvis i aorta ring assay. Vi foreslår, at aorta ring analysen kan bruges til at studere angiogene potentiale for enhver fysisk støtte celletype når under efterforskning for angiogene regenerativ applikationer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Alpha-MEM</td>
			<td >Gibco</td>
			<td >12571071</td>
			<td >For FTM HUCPVC and BMSC culture media.</td>
		</tr>
		<tr >
			<td >APC-conjugated anti human TRA-1-85</td>
			<td >R&#38;D Systems</td>
			<td >FAB3195A</td>
			<td >Human-specific cell marker for flow cytometry and cell sorting</td>
		</tr>
		<tr >
			<td >Basal membrane extract (BME) (Matrigel)</td>
			<td >Corning</td>
			<td >354234</td>
			<td >For aorta embedding</td>
		</tr>
		<tr >
			<td >Bullet-kit</td>
			<td >Lonza</td>
			<td >CC-3162</td>
			<td >Includes: Gentamicin/Amphotericin-B 
			(GA)human Epidermal 
			Growth Factor (hEGF); Vascular 
			Endothelial Growth Factor (VEGF); R3- 
			Insulin-like Growth Factor-1 (R3-IGF-1); 
			Ascorbic Acid; Hydrocortisone; 
			human Fibroblast Growth Factor-Beta (hFGF-&#946;); Heparin; Fetal Bovine Serum 
			(FBS). Required to prepare EGM</td>
		</tr>
		<tr >
			<td >CellTracker Green CMFDA Dye</td>
			<td >Thermo-fisher</td>
			<td >C2925</td>
			<td >For staining MSCs, green is picked up optimally by MSCs</td>
		</tr>
		<tr >
			<td >CKX53 Culture Microscope</td>
			<td >Olympus</td>
			<td ></td>
			<td >For bright-field imaging of endothelial network development</td>
		</tr>
		<tr >
			<td >Countess automated cell counter</td>
			<td >Invitrogen</td>
			<td >C10227</td>
			<td >Cell counting for MSC culture, flow cytometry and qPCR</td>
		</tr>
		<tr >
			<td >Dispase</td>
			<td >StemCell technologies</td>
			<td >7923</td>
			<td >For dissociating aortic ring-MSC co-cultures (pre-warm at 37 &#176;C)</td>
		</tr>
		<tr >
			<td >Disposable sterile scalpels</td>
			<td >VWR</td>
			<td >21909-654</td>
			<td >For sectioning aorta</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s phosphate buffered saline</td>
			<td >Sigma-Aldrich</td>
			<td >D8537</td>
			<td >PBS. 1X, Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr >
			<td >Endothelial basal media (EBM)</td>
			<td >Lonza</td>
			<td >CC-3156</td>
			<td >Basal media required for culturing aortic ring assay-MSC co-cultures (warm at 37 &#176;C before use). Required for EGM and EBM-FBS</td>
		</tr>
		<tr >
			<td >Ethanol, 70%, Biotechnology Grade</td>
			<td >VWR</td>
			<td >97064-768</td>
			<td >To sterilize surfaces</td>
		</tr>
		<tr >
			<td >EVOS</td>
			<td >Life Technologies</td>
			<td ></td>
			<td >In-house fluorescent microscope to track MSC migration and integration</td>
		</tr>
		<tr >
			<td >Fetal bovine serum (FBS) (Hyclone)</td>
			<td >GE Healthcare</td>
			<td >SH3039603</td>
			<td >Serum component of cell culture medium</td>
		</tr>
		<tr >
			<td >FITC-conjugated anti-CD31 antibody</td>
			<td >BD</td>
			<td >558068</td>
			<td >Human endothelial marker for flow cytometry</td>
		</tr>
		<tr >
			<td >FITC-conjugated anti-CD146antibody</td>
			<td >BD</td>
			<td >560846</td>
			<td >Human pericyte marker for flow cytometry</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >Almedic</td>
			<td >7727-A10-704</td>
			<td >For handing rat tissue. Can use any similar forceps</td>
		</tr>
		<tr >
			<td >Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td >Life Technologies</td>
			<td >14175-094</td>
			<td >1X Without calcium chloride and magnesium chloride</td>
		</tr>
		<tr >
			<td >HERAcell 150i CO2 Incubator</td>
			<td >Thermo Fisher Scientific</td>
			<td >51026410</td>
			<td >For incubating cells</td>
		</tr>
		<tr >
			<td >Human Angiogenesis RT2 profiler PCR array</td>
			<td >Qiagen</td>
			<td >PAHS-024Z</td>
			<td >Human specific and includes primers for 84 genes involved in angiogenesis. Each well is 1 primer reaction</td>
		</tr>
		<tr >
			<td >ImageJ</td>
			<td ></td>
			<td ></td>
			<td >Open source image processing software. Require Angiogenesis analyzer plugin</td>
		</tr>
		<tr >
			<td >LSR II</td>
			<td >BD</td>
			<td ></td>
			<td >UHN SickKids FC Facility. For flow cytometry.</td>
		</tr>
		<tr >
			<td >MoFlo Astrios</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td >UHN SickKids FC Facility. For cell sorting.</td>
		</tr>
		<tr >
			<td >Penicillin/streptomycin</td>
			<td >Gibco</td>
			<td >15140122</td>
			<td >Antibiotic component to buffers and cell culture medium</td>
		</tr>
		<tr >
			<td >RNeasy Mini Kit</td>
			<td >Qiagen</td>
			<td >74104</td>
			<td >For RNA purification. Includes cell lysis buffer</td>
		</tr>
		<tr >
			<td >RT2Easy First Strand Kit</td>
			<td >Qiagen</td>
			<td >330421</td>
			<td >For preparation of cDNA for qPCR</td>
		</tr>
		<tr >
			<td >RT2PreAMP cDNA Synthesis Kit</td>
			<td >Qiagen</td>
			<td >330451</td>
			<td >Pre-amplification of cDNA if low-yield RNA</td>
		</tr>
		<tr >
			<td >Surgical scissors</td>
			<td >Fine Science Tools</td>
			<td >14059-11</td>
			<td >For cutting skin, muscle and aorta</td>
		</tr>
		<tr >
			<td >Sterile gauze</td>
			<td >VWR</td>
			<td >3084</td>
			<td >To dampen and sterilize chest fur</td>
		</tr>
		<tr >
			<td >TrypleE</td>
			<td >Thermo Fisher Scientific</td>
			<td>12605036</td>
			<td >MSC dissociation enzyme pre-warm at 37 &#176;C</td>
		</tr>
		<tr >
			<td >0.2 &#956;m pore filtration unit</td>
			<td >Thermo Fisher Scientific</td>
			<td >566-0020</td>
			<td >To sterilize tissue culture media</td>
		</tr>
		<tr >
			<td >0.25% Trypsin/EDTA</td>
			<td >Gibco</td>
			<td >25200056</td>
			<td >For cell dissociation, pre-warm at 37 &#176;C</td>
		</tr>
		<tr >
			<td >10 cm tissue culture dishes</td>
			<td >Corning</td>
			<td >25382-428</td>
			<td >For cleaning and sectioning aorta and MSC cell culture</td>
		</tr>
		<tr >
			<td >12 well-cell culture plates</td>
			<td >Corning-Sigma Aldrich</td>
			<td >CLS3513</td>
			<td >For setting up aortic ring assay-MSC co-cultures</td>
		</tr>
		<tr >
			<td >15 mL tube</td>
			<td >BD Falcon</td>
			<td >352096</td>
			<td >For general tissue culture procedures</td>
		</tr>
		<tr >
			<td >70 &#956;m cell strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td >To ensure a single cell suspension before flow cytometry or sorting</td>
		</tr>
	</tbody>
</table>

the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro

Angiogenese er en kompleks, stærkt reguleret proces, der er ansvarlig for at levere og vedligeholde passende væv perfusion. Utilstrækkelig Vaskulaturen vedligeholdelse og patologiske misdannelser kan resultere i alvorlige iskæmiske sygdomme, mens alt for rigelige vaskulære udvikling er forbundet med kræft og inflammatoriske lidelser. En lovende form af pro-angiogene terapi er brugen af angiogene celler kilder, der kan levere såvel regulerende faktorer som fysisk støtte for nyligt udvikle Vaskulaturen.
Mesenchymale stromale celler (MSCs) er udførligt undersøgte kandidater til vaskulær regeneration på grund af deres paracrine virkninger og deres evne til at opdage og hjemsted for iskæmisk eller betændt væv. Især første trimester menneskelige navlestrengen perivascular celler (FTM HUCPVCs) er en meget lovende kandidat på grund af deres pericyte-lignende egenskaber, høj proliferativ og multilineage potentiale, immun-privilegeret egenskaber og robust paracrine profil. Effektivt evaluering potentielt angiogene regenerativ celler, er det en forudsætning at teste dem i pålidelige og "oversættes" præ-klinisk assays. Aorta ring assay er en ex vivo angiogenese model, der giver mulighed for nem kvantificering af rørformede endotel strukturer, indeholder tilbehør støttende celler og ekstracellulære matrix (ECM) fra værten, udelukker inflammatoriske komponenter, og er hurtig og billig at etablere. Dette er fordelagtigt i forhold til i vivo modeller (fx, cornea assay, Matrigel plug assay); aorta ring assay kan spore de administrerede celler og observere intercellulære interaktioner samtidig undgå xeno-immune afvisning.
Vi præsenterer en protokol for en roman anvendelse af aorta ring assay, som omfatter menneskelige MSCs i co kulturer med udviklingslandene rotte aorta endotel netværk. Denne analyse giver mulighed for analyse af MSC bidrag til tube dannelse og udvikling gennem fysisk pericyte-lignende interaktioner og deres styrke til aktivt overflytning til websteder af angiogenese, og for at vurdere deres evne til at udføre og mægle ECM behandling. Denne protokol giver yderligere oplysninger om ændringer i MSC fænotype og gen udtryk efter fælles kultur.

Den skematiske arbejdsgang for oprettelse af aorta ring/MSC Co kultur assay er vist i figur 1. De vigtigste skridt omfatter: rotte aorta isolation, skæring og indlejring af aorta ringene, overvågning af endotel spiring og udvikling af net, og endelig mærkning og administration af MSCs. Tidslinjen i endothelial netværksanalyse skitserer vinduet for de analyser, der er muligt for hver periode på Co dyrkning: dag 1, 5 &#38; 7. Yderligere noter er fremhævet...

Watch this Scientific Journal Video about The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro at JoVE.com

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Her præsenterer vi en roman anvendelse af aorta ring assay hvor prelabelled mesenchymale celler er co kulturperler med rotte aorta-afledte endotel netværk. Denne nye metode giver mulighed for visualisering af mesenchymale stromale celler (MSCs) målsøgende og integration med endotel netværk, kvantificering af Netværksegenskaber og evaluering af MSC immunophenotypes og gen udtryk.

Aorta Ring Co kultur Assay: Et praktisk værktøj til at vurdere angiogene af mesenchymale stromale celler In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

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Developmental Biology

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

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Induktion af Mesenchymale-Epithelial Overgange i sarkomceller

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

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