Name: in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse
Uploaded: 2016-12-19T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse Video at JoVE.com

Dr. K. Yuan was supported by an American Heart Association Scientist Development Grant (15SDG25710448) and the Pulmonary Hypertension Association Proof of Concept Award (SPO121940). Dr. V. de Jesus Perez was supported by a career development award from the Robert Wood Johnson Foundation, an NIH K08 HL105884-01 award, a Pulmonary Hypertension Association Award, a Biomedical Research Award from the American Lung Association and a Translational Research and Applied Medicine award from Stanford University.

Etik Statement: Procedurer, der involverer dyr fag er blevet godkendt af Institutional Animal Care og brug Udvalg ved Stanford University School of Medicine. BEMÆRK: Dyr er under bedøvelse med 3% vaporizer isofluran og 3% forsyning af O 2 gas. Anvendelse af dyrlæge salve på øjnene kan medvirke til at forebygge tørhed, mens under anæstesi. 1. Cell Fremstilling <ol><li> Grow menneskelige endotel og pericyt cellekulturer i 100 mm plader med 10 ml af den relevante medier. Erstat 10 ml fri...

<ol>
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Matrixgelen plug assay har vist sig at være en bekvem og effektiv metode til at evaluere genregulering i angiogenese, angiogene og antiangiogene forbindelser in vivo, og at supplere in vitro-tests. Her beskriver vi detaljeret en hidtil ukendt matrix gel plug-assay af human angiogenese der undersøger interaktionen mellem endotelceller og pericytter under dannelsen fartøj. Der er et par nye og kritiske skridt i denne protokol. Celler i lav passage (passage 1 - 4) er at for...

Angiogenese er den proces, hvor nye blodkar dannes ud fra en allerede eksisterende vaskulære netværk 1 og er fokus for igangværende forskning på tværs af mange områder, der spænder fra normal udvikling til sygdom. Denne dynamiske proces involverer proliferation og migration af endotelceller (ECS) og rekruttering af pericytter at konstruere en vaskulær rør, der er rettet mod det websted, der har brug for ilt og næringsstoffer levering 2. For at undersøge denne proces kræver en lige så dynamisk assay, vigtigst en, der kan rekapitulere den tredimensionale karakter dannelse rør. In vitro er blevet udviklet 3D matrix-analyser for at løse dette behov og har fungeret godt at give forskerne at definere de diskrete trin i tid og rum, hvor angiogenese finder sted 3, 4, 5, 6. Imidlertid er disse in vitro 3D matrix-modeller begrænset til at studere ikke-perfunderede fartøjer and mangler derfor kritiske komponenter er relevante for angiogenese processen (fx cirkulerende vækst og hæmmende faktorer, unaturlige spændinger / kræfter på tværs af vaskulære seng) og undlader at simulere komplekse miljø til stede i levende væv. For at løse denne begrænsning har flere in vivo angiogenese analyser er udviklet 7, herunder matrix gel plug assay, som vil være i fokus i vores rapport 8, 9. Matricen gel plug-analysen er en veletableret in vivo angiogenese assay, der appellerer til forskere, da det giver en robust platform til at teste roller forskellige celler og stoffer i angiogenese. Matrixgel er en kommercielt tilgængelig basalmembran løsning, der udskilles af Engelbreth-Holm-Swarm (EHS) muse sarcoma-cellelinie som størkner til en gel-lignende materiale ved 37 ° C. Matrixgelen kan blandes med celler og / eller stoffer, såsom vækstfaktorer og InjeCTED subkutant i mus. Værten EC&#39;er vil invadere proppen over 14 dage, danner en vaskulære netværk, og blive perfunderet med værtens blod. Til dato har matrix gel plug analyser udelukkende fokuseret på studiet af endotelceller opførsel under angiogenese dog til vores bedste overbevisning er der endnu ikke gjort noget for at afgøre, om denne analyse kan bruges til at co-kultur endotelceller og pericytter at undersøge, hvordan disse to celletyper interagerer under angiogenese. Specifikt forstå forholdet mellem EC&#39;er og pericytes er værdifuld til undersøgelse af sygdomme, hvor tab blodkar er patologisk, herunder mikrovaskulære iskæmi og perifer karsygdom 10, 11, 12. Her beskriver vi en protokol, der introducerer human-afledte pericytter til matricen gel blandingen sammen med human EC&#39;er og fibroblastvækstfaktor (bFGF). Denne blanding kan derefter injiceres subkutant in dorsum af SCID-mus at tillade dannelse af fuldt funktionelle, pericyt overtrukket, hybride fartøjer. Vores protokol beskriver, hvordan man forbereder matrix gel propper indeholdende humane EC&#39;er enten med eller uden menneskelige pericytter, placering i SCID-mus og hvordan man kan analysere de histologiske snit til kritiske angiogenese endepunkter.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">PBS (Phosphate buffered saline)</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.25% Trypsin/0.53mM EDTA</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Endothelial Cell Media (ECM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1001</td>
			<td>includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Pericyte Media (PM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1201</td>
			<td>includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary microvascular endothelial cells</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is also available commercially. Cells used at passage 1-4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary pericytes</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is not available commercially, but brain paricytes are.&#160; Cells used at passage 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">bFGF (basic Fibroblast Growth Factor)</td>
			<td style="width:115px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>stock solution is 50ug/ml in 0.1% BSA in PBS, aliquots at 50 uL and stored at -20 degrees</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">356237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">28G 1cc Insluin Syringe</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">329410</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">SCID (Severe Combined Immune Deficiency) mice</td>
			<td style="width:115px;">The Jackson Laboratory</td>
			<td>5557</td>
			<td>NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">1.5mL microcentrifuge tubes, sterile</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">15 mL screw top tubes, sterile</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">PAP pen</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">8899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">hemocytometer</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Nair hair removal cream</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">anti-human CD31 primary antibody</td>
			<td style="width:115px;">LifeSpan Biosciences</td>
			<td>LS-B4737</td>
			<td>working solution is 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human Smooth Muscle actin CY3 primary fluorescent antibody</td>
			<td style="width:115px;">Sigma</td>
			<td>C6198</td>
			<td>working solution is 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit secondary antibody; 488/green</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A-11008</td>
			<td>working solution is 1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Prolong Gold DAPI solution</td>
			<td style="width:115px;">Cell Signaling</td>
			<td style="width:119px;">8961S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">microscope slides</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">48300-047</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">no. 1.5 cover slips</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-544-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">citrate buffer 10x</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">21545</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">extra fine surgical scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Formalin (paraformaldehyde)</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">245-685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tissue cassettes</td>
			<td style="width:115px;">Simport</td>
			<td>M492-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">goat serum</td>
			<td style="width:115px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

Repræsentant H &amp; E og immunfluorescensfarvning af matrix gel plug sektioner er vist i figur 2. Sektioner fra EF kun stikpropper vise nogle fartøjer, der er for det meste ikke er perfunderet med blod (Figur 2 øverst til venstre, sorte pile), mens propper indeholder både ECS pericyter viser flere perfunderet fartøjer med større diametre og fulde pericyt dækning, som det fremgår af positiv SMA farvning umiddelbart op til CD31-positive EC&#39;er....

Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse Video at JoVE.com

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse Video (Video) | JoVE

Vi præsenterer en protokol til at studere human endotel-pericyt interaktioner i mus ved anvendelse af en variation af matricen gel plug angiogenese assay.

In vivo studie af human endotel-pericyt Interaktion Brug af Matrix Gel Plug Assay i Mus

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

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In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

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