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 taknemmelige for den fremragende teknisk bistand fra Janine Becker, Caroline Bickelmann og Ruth Nickels. Denne undersøgelse blev finansieret af en bevilling på Deutsche Forschungsgemeinschaft (DFG - German Research Foundation) - LA 2682 / 7-1.

Alle procedurer blev udført i overensstemmelse med National Institute of Health retningslinjer for brugen af ​​forsøgsdyr og fulgte institutionelle retningslinier (Landesamt für Soziales, Gesundheit und Verbraucherschutz, Abt. Lebensmittel- und Veterinärwesen, Zentralstelle, Saarbrücken, Tyskland). 1. Fremstilling af kirurgiske instrumenter <ol><li> Holde klar dissektion saks, kirurgiske pincet, små forberedelse saks, fine pincet og en steril petriskål med 15 ml Dulbeccos modifi...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+and+cytochemical+localization+of+a+catecholamine-sensitive+adenylate+cyclase+in+isolated+capillary+endothelium.">Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium.</a> Proc Natl Acad Sci U S A. 69 (11), 3175-3179 (1972).</li><li>Wagner, R. C., Matthews, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+culture+of+capillary+endothelium+from+epididymal+fat.">The isolation and culture of capillary endothelium from epididymal fat.</a> Microvasc Res. 10 (3), 286-297 (1975).</li><li>Laschke, M. W., Menger, M. 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I denne undersøgelse præsenterer vi en veletableret protokol til isolering af ad-MVF. Indhentning ad-MVF fra murine fedtvæv er en enkel procedure med et par kritiske trin. Mus udviser forskellig subkutan og intraabdominale fedtdepoter. Som tidligere beskrevet for rotter, den mest egnede fedtkilde til isolering af ad-MVF er epididymisfedtpuderne grund af deres størrelse, homogen struktur og minimal kontaminering med større blodkar 11, 12. I modsætning herti...

Tissue engineering fokuserer på fremstillingen af vævs- og organ-erstatninger, der opretholder, genoprette eller forøge funktionen af inoperabel in vivo modstykker 1, 2. Den skæbne manipuleret væv konstruktioner er helt afhængig af en tilstrækkelig vaskularisering 3. Mikrovaskulære netværk inden disse konstruktioner skal hierarkisk organiseret med arteriolerne, kapillærer og små blodårer til at tillade effektiv blod perfusion efter inosculation til modtagerens kar 4. Dannelsen af ​​sådanne netværk er blandt de centrale udfordringer i tissue engineering. Til dette formål har et bredt spektrum af eksperimentelle vaskularisering strategier blevet indført i løbet af de sidste to årtier 5, 6. Angiogene fremgangsmåder stimulerer indvæksten af ​​modtagende mikrokar i manipulerede tissdier ved hjælp af strukturel eller fysisk stillads modifikation, såsom inkorporering af vækstfaktorer 7. Men for vaskularisering af store tredimensionale konstruktioner, angiogeneseafhængige strategier er markant begrænset af langsomme vækstrater for udvikling af mikrokar 8. Derimod begrebet prevascularization sigter til generering af funktionelle mikrovaskulære netværk inden væv konstruerer før deres implantation 9. Konventionel prevascularization involverer co-dyrkning af fartøjers-dannende celler, såsom endotelceller, murkrone celler eller stamceller 10, inden stilladser. Efter mikrovaskulær netværksdannelse kan de prevascularized konstruktioner derefter implanteres i vævsdefekter. Bemærkelsesværdige, denne prevascularization tilgang er vanskelig at anvende i et klinisk miljø, fordi den er baseret på komplekse og tidskrævende in vitro &lt;/ em&gt; procedurer, som er begrænset af de store lovgivningsmæssige forhindringer 9. Følgelig er der stadig et behov for udviklingen af ​​hidtil ukendte prevascularization strategier, der er mere egnet til en bred klinisk anvendelse. En sådan prevascularization strategi kan være anvendelsen af ​​adipøst væv-afledte mikrovaskulære fragmenter (ad-MVF). ad-MVF repræsenterer potente vaskularisering enheder, som kan høstes i store mængder fra fedtvævet af rotter 11, 12 og mus 13. De består af arteriolær, kapillær, og venular karsegmenter, der udviser en fysiologisk mikrokar morfologi med et lumen og stabiliserende perivaskulære celler 14, 15. Denne unikke funktion giver den umiddelbare implantation af ad-MVF-seedede stilladser i væv defekter uden precultivation. Der er ad-MVF hurtigt samle itil funktionelle mikrovaskulære netværk. Endvidere ad-MVF repræsenterer en rig kilde til mesenkymale stamceller 16, som yderligere kan bidrage til deres slående regenerationsevne. Følgelig er ad-MVF stigende grad anvendes i forskellige områder af vævsmanipulering 14, 15, 17, 18, 19, 20, 21. Isoleringen af ad-MVF oprindeligt blev etableret i rotter 11, 12. Heri beskriver vi en protokol, der tillader den standardiserede isolering af henholdsvis murine ad-MVF fra epididymale fedtpuder. Dette kan give yderligere indsigt i molekylære mekanismer bag ad-MVF funktion ved hjælp af 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 funktionelt mikrovaskulær netværk er af central betydning for overlevelse og integration af manipulerede vævskonstruktioner. Til dette formål er der etableret flere angiogene og prevascularization strategier. Men de fleste cellebaserede midler indbefatter tidskrævende in vitro trin til dannelse af en mikrovaskulær netværk. Derfor er de ikke egnede til intraoperative éttrins procedurer. Adipøst vævsafledte mikrovaskulære fragmenter (ad-MVF) repræsenterer lovende vaskularisering enheder. De let kan isoleres fra fedtvæv og udviser en funktionel mikrokar morfologi. Desuden har de hurtigt samle ind i nye mikrovaskulære netværk efter in vivo implantation. Desuden har ad-MVF vist sig at inducere lymfangiogenese. Endelig er de en rig kilde til mesenkymale stamceller, som yderligere kan bidrage til deres høje vaskularisering potentiale. I tidligere undersøgelser har vi vist den bemærkelsesværdige vascularizatipå kapaciteten af ​​ad-MVF i manipuleret knogle og hud erstatninger. I den foreliggende undersøgelse rapporterer vi på en standardiseret protokol for den enzymatiske isolation af ad-MVF fra murine fedtvæv.

I den foreliggende undersøgelse har vi udført seks ad-MVF isoleringsprocedurer med fedtvæv fra 7- til 12-måneder gammel mand vildtype C57BL / 6-mus (gennemsnitlig kropsvægt: 35 ± 1 g). Figur 1 illustrerer høst af murine epididymisfedtpuderne med efterfølgende mekanisk og enzymatisk ad-MVF isolation. Den nødvendige tid for høstning af fedt var 30 minutter, og til isolering af ad-MVF var 120 min. I alt proceduren tog 150 min. <p class="jove_content" fo:keep-t...

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 præsenterer en protokol til isolering fedtvævslidelser-afledte mikrovaskulære fragmenter, der repræsenterer lovende vaskularisering enheder. De kan hurtigt isoleres, ikke kræver in vitro behandling og således kan anvendes til et-trins prevascularization i forskellige områder af vævsmanipulering.

Isolering af murine fedtvæv-afledte mikrovaskulær Fragmenter som Vaskularisering Enheder til Tissue Engineering

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

Research

JoVE Journal

Bioengineering

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