Name: isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering
Uploaded: 2017-04-30T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

Vi er takknemlige for utmerket teknisk assistanse av Janine Becker, Caroline Bickelmann og Ruth Nickels. Denne studien ble finansiert av et stipend fra Deutsche Forschungsgemeinschaft (DFG - Tysk Research Foundation) - LA 2682 / 7-1.

Alle prosedyrer ble utført i henhold til National Institute of Health retningslinjer for bruk av forsøksdyr og fulgt institusjonelle retningslinjer (Landesamt für Soziales, Gesundheit und Verbraucherschutz, Abt. Lebensmittel- und Veterinärwesen, Zentralstelle, Saarbrücken, Tyskland). 1. Fremstilling av kirurgiske instrumenter <ol><li> Hold klar disseksjon saks, kirurgisk tang, små fremstillings saks, fin pinsett og en steril petriskål med 15 ml Dulbeccos modifiserte Eagle medium (DM...

<ol>
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I denne studien presenterer vi en godt etablert protokoll for isolering av ad-MVF. Innhenting av ad-MVF fra murine fettvev er en enkel prosedyre med noen kritiske trinn. Mus som utviser forskjellig subkutan og intraabdominale fettdepoter. Som tidligere beskrevet for rotter, mest egnet fettkilde for isolering av ad-MVF er de epididymale fettputer på grunn av sin størrelse, homogen struktur og minimal forurensning med større blodkar 11, 12. I motsetning til det...

Tissue teknikk fokuserer på fremstilling av vevs- og organ substitutter som opprettholder, restaurere eller forsterke funksjonen av inoperable in vivo motstykker 1, 2. Skjebnen av konstruerte vev konstruksjoner avgjørende avhengig av tilstrekkelig vaskularisering tre. Mikrovaskulære nettverk innen disse konstruksjonene bør hierarkisk organisert med arterioler, kapillærer og venules å tillate effektiv blod perfusjon etter inosculation til mottakerens blodkar 4. Generering av slike nettverk er blant de viktigste utfordringene i tissue engineering. For dette formålet, har et bredt spekter av eksperimentelle vaskularisering strategier blitt introdusert i løpet av de siste to tiårene 5, 6. Angiogene tilnærminger stimulere innvekst av mottaker microvessels inn konstruert tissues ved hjelp av strukturell eller fysikalsk-kjemisk modifikasjon stillas, slik som inkorporering av vekstfaktorer til 7. Men for vaskularisering av store tredimensjonale konstruksjoner, angiogenese-avhengige strategier er markert begrenset av lave vekstrater for å utvikle microvessels 8. I motsetning til dette begrepet prevascularization mål for frembringelse av funksjonelle mikrovaskulære nettverk innen vev konstruerer før implantering 9. Konvensjonell prevascularization omfatter ko-kulturen av fartøy-dannende celler, slik som endotelceller, celler eller vegg stamceller 10, innen stillasene. Etter at mikrovaskulær nettverkdannelse, kan de prevascularized konstruksjonene deretter implanteres i vevsdefekter. Verdt å merke seg, er dette prevascularization tilnærmingen vanskelig å anvende i en klinisk setting, fordi den er basert på komplekse og tidkrevende in vitro &lt;/ em&gt; prosedyrer, som er begrenset av store regulatoriske hindringer 9. Følgelig er det fortsatt behov for utvikling av nye prevascularization strategier som er mer egnet for en bred klinisk anvendelse. En slik strategi kan være prevascularization anvendelsen av adipose tissue-avledet mikrovaskulære fragmenter (ad-MVF). ad-MVF representere potent vaskularisering heter som kan høstes i store mengder fra fettvev hos rotter 11, 12 og 13 mus. De består av arteriolar, kapillær, og venular kar-segmenter, som oppviser en fysiologisk microvessel morfologi med en lumen og stabiliserende perivaskulære celler 14, 15. Denne unike egenskap tillater den umiddelbare implantasjon av ad-MVF foret stillasene inn i vevsdefekter uten precultivation. Der ad-MVF raskt montere itil funksjonelle mikrovaskulære nettverk. Videre ad-MVF representere en rik kilde av stamceller 16, som i tillegg kan bidra til deres slående evne til reproduksjon. Følgelig er ad-MVF stadig mer brukt i ulike områder av vevsteknologi 14, 15, 17, 18, 19, 20, 21. Isoleringen av ad-MVF har opprinnelig blitt fastslått i rotter 11, 12. Heri beskriver vi en protokoll som gjør det mulig standardisert isolering av muse-ad-MVF fra epididymalvekt fett pads. Dette kan gi ytterligere innsikt i molekylære mekanismer som ligger til grunn ad-MVF funksjon ved anvendelse av transgene musemodeller.

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	<tbody>
		<tr>
			<td>1.5-mL conical microcentrifuge tube</td>
			<td>VWR, Kelsterbach, Germany</td>
			<td>700-5239</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;L precision pipette</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>4920000059</td>
			<td></td>
		</tr>
		<tr>
			<td>10-mL measuring pipette</td>
			<td>Costar, Corning Inc., New York, USA</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>14-mL PP tubes</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>187261</td>
			<td></td>
		</tr>
		<tr>
			<td>1-mL precision pipette</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr>
			<td>500-&#181;m filter (pluriStrainer 500 &#181;m)</td>
			<td>HISS Diagnostics, Freiburg, Germany</td>
			<td>43-50500-03</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical centrifuge tube</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL Erlenmeyer flask</td>
			<td>VWR, Kelsterbach, Germany</td>
			<td>214-0211</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>65518</td>
			<td></td>
		</tr>
		<tr>
			<td>cell detachment solution (Accutase)</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>00-4555-56</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River, Cologne, Germany</td>
			<td>027</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6-Tg(CAG-EGFP)1Osb/J mice</td>
			<td>The Jackson Laboratory, Bar Harbor, USA</td>
			<td>003291</td>
			<td></td>
		</tr>
		<tr>
			<td>CD117-FITC</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31-PE</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553354</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase NB4G&#160;</td>
			<td>Serva Electrophoresis GmbH, Heidelberg, Germany</td>
			<td>17465.02</td>
			<td>Lot tested by manufacturer for enzymatic activity</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Braun Aesculap AG &#38;CoKG, Melsungen, Germany</td>
			<td>BC 601</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-binding dye (Bisbenzimide H33342)</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)&#160;</td>
			<td>PAN Biotech, Rickenbach, Germany</td>
			<td>P04-03600</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Biochrom GmbH, Berlin, Germany</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>S&#38;T AG, Neuhausen, Switzerland</td>
			<td>FRS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>World Precision Instrumets, Sarasota, FL, USA</td>
			<td>503261</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermal skin substitute (Integra)</td>
			<td>Integra Life Sciences, Sain Priest, France</td>
			<td>62021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Serumwerk Bernburg AG, Bernburg, Germany</td>
			<td>7005294</td>
			<td></td>
		</tr>
		<tr>
			<td>M-IgG2akAL488&#160;&#160;</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>53-4724-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Octeniderm (disinfecting solution)</td>
			<td>Sch&#252;lke &#38; Mayer, Norderstedt, Germany</td>
			<td>118211</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>A2213</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner bio-one, Frickenhausen, Germany</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Lonza Group, Basel, Switzerland</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 20-&#181;m (20 &#181;m filter)</td>
			<td>HISS Diagnostics, Freiburg, Germany</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat-IgG2akFITC</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553988</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat-IgG2akPE</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>553930</td>
			<td></td>
		</tr>
		<tr>
			<td>Small preparation scissors</td>
			<td>S&#38;T AG, Neuhausen, Switzerland</td>
			<td>SDC-15 R-8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>Braun Aesculap AG &#38;CoKG, Melsungen, Germany</td>
			<td>BD510R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape (Heftpflaster Seide) 1.25 cm</td>
			<td>Fink &#38; Walter GmbH, Mechweiler, Germany</td>
			<td>1671801</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine&#160;</td>
			<td>Bayer Vital GmbH, Leverkusen, Germany</td>
			<td>1320422</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-SMA-AL488</td>
			<td>eBioscience, San Diego, CA USA</td>
			<td>53-9760-82</td>
			<td>Intracellular labeling additionally requires Cytofix/Cytoperm (BD Biosciences, Heidelberg, Germany; #554722)</td>
		</tr>
	</tbody>
</table>

isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering

Et funksjonelt mikrovaskulær nettverk er av dreibar betydning for overlevelse og integrering av konstruerte vev konstruksjoner. For dette formålet, er det etablert flere angiogene og prevascularization strategier. Imidlertid er de fleste cellebaserte tilnærminger inkluderer tidkrevende in vitro fremgangsmåten for dannelse av en mikrovaskulær nettverk. Derfor, er de ikke egnet for intra ett-trinnsprosedyrer. Adipose tissue-avledet mikrovaskulære fragmenter (ad-MVF) representerer lovende vaskularisering enheter. De kan lett isoleres fra fettvevet og utviser en funksjonell microvessel morfologi. Videre må de raskt montere inn nye mikrovaskulære nettverk etter implantering in vivo. I tillegg har ad-MVF vist å indusere lymphangiogenesis. Til slutt, de er en rik kilde av mesenchymale stamceller, som ytterligere kan bidra til deres høye vaskularisering potensial. I tidligere studier har vi vist bemerkelsesverdig vascularizatipå kapasitet på ad-MVF i konstruerte bein og hud substitutter. I den foreliggende undersøkelse rapporterer vi på en standardisert protokoll for den enzymatiske isolering av ad-MVF fra murine fettvev.

I den foreliggende undersøkelse utført vi seks ad-MVF isoleringsprosedyrer med fettvev fra 7- til 12-måneder gamle hann villtype C57BL / 6-mus (gjennomsnittlig kroppsvekt: 35 ± 1 g). Figur 1 illustrerer den høsting av murine epididymal fettputer, med etterfølgende mekanisk og enzymatisk ad-MVF isolasjon. Den tid som er nødvendig for innhøsting av fett var 30 minutter og for isolering av ad-MVF var 120 min. Totalt ble prosedyren tok 150 min. <p class="jove_con...

Watch this Scientific Journal Video about Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering at JoVE.com

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Vi presenterer en protokoll for å isolere fettvevs-avledet mikrovaskulære fragmenter som representerer lovende vaskularisering enheter. De kan isoleres hurtig, ikke krever in vitro bearbeiding, og således kan anvendes for ett-trinns prevascularization innenfor forskjellige vev engineering.

Isolering av Murine fettvev-avledet mikrovaskulær Fragmenter som vaskularisering Enheter for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

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In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

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The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...