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

Nous sommes reconnaissants pour l&#39;excellente assistance technique de Janine Becker, Caroline Bickelmann et Ruth Nickels. Cette étude a été financée par une subvention de la Deutsche Forschungsgemeinschaft (DFG - Fondation allemande pour la recherche) - LA 2682 / 7-1.

Toutes les procédures ont été effectuées selon l&#39;Institut national des directives de santé pour l&#39;utilisation des animaux de laboratoire et ont suivi les directives institutionnelles (Landesamt für Soziales, Gesundheit und Verbraucherschutz, Abt. Lebensmittel- und Veterinärwesen, Zentralstelle, Sarrebruck, Allemagne). 1. Préparation des instruments chirurgicaux <ol><li> Tenir prêt les ciseaux de dissection, des pinces chirurgicales, des petits ciseaux de préparation, une ...

<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=Vascularization+is+the+key+challenge+in+tissue+engineering.">Vascularization is the key challenge in tissue engineering.</a> Adv Drug Deliv Rev. 63 (4-5), 300-311 (2011).</li><li>Rouwkema, J., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+and+Angiogenesis+in+Tissue+Engineering:+Beyond+Creating+Static+Networks.">Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks.</a> Trends Biotechnol. 34 (9), 733-745 (2016).</li><li>Laschke, M. W., Menger, M. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevascularization+in+tissue+engineering:+Current+concepts+and+future+directions.">Prevascularization in tissue engineering: Current concepts and future directions.</a> Biotechnol Adv. 34 (2), 112-121 (2016).</li><li>Baiguera, S., Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelialization+approaches+for+viable+engineered+tissues.">Endothelialization approaches for viable engineered tissues.</a> Angiogenesis. 16 (1), 1-14 (2013).</li><li>Wagner, R. C., Kreiner, P., Barrnett, R. J., Bitensky, 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=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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+microvascular+fragments:+natural+vascularization+units+for+regenerative+medicine.">Adipose tissue-derived microvascular fragments: natural vascularization units for regenerative medicine.</a> Trends Biotechnol. 33 (8), 442-448 (2015).</li><li>Laschke, M. W., 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=Vascularisation+of+porous+scaffolds+is+improved+by+incorporation+of+adipose+tissue-derived+microvascular+fragments.">Vascularisation of porous scaffolds is improved by incorporation of adipose tissue-derived microvascular fragments.</a> Eur Cell Mater. 24, 266-277 (2012).</li><li>Frueh, F. S., 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=Adipose+tissue-derived+microvascular+fragments+improve+vascularization,+lymphangiogenesis+and+integration+of+dermal+skin+substitutes.">Adipose tissue-derived microvascular fragments improve vascularization, lymphangiogenesis and integration of dermal skin substitutes.</a> J Invest Dermatol. 137 (1), 217-227 (2017).</li><li>McDaniel, J. S., Pilia, M., Ward, C. L., Pollot, B. E., Rathbone, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+multilineage+potential+of+cells+derived+from+isolated+microvascular+fragments.">Characterization and multilineage potential of cells derived from isolated microvascular fragments.</a> J Surg Res. 192 (1), 214-222 (2014).</li><li>Nakano, 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=Effect+of+autotransplantation+of+microvessel+fragments+on+experimental+random-pattern+flaps+in+the+rat.">Effect of autotransplantation of microvessel fragments on experimental random-pattern flaps in the rat.</a> Eur Surg Res. 30 (3), 149-160 (1998).</li><li>Nakano, 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=Successful+autotransplantation+of+microvessel+fragments+into+the+rat+heart.">Successful autotransplantation of microvessel fragments into the rat heart.</a> Eur Surg Res. 31 (3), 240-248 (1999).</li><li>Shepherd, B. R., Hoying, J. B., Williams, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+transplantation+after+acute+myocardial+infarction.">Microvascular transplantation after acute myocardial infarction.</a> Tissue Eng. 13 (12), 2871-2879 (2007).</li><li>Pilia, 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=Transplantation+and+perfusion+of+microvascular+fragments+in+a+rodent+model+of+volumetric+muscle+loss+injury.">Transplantation and perfusion of microvascular fragments in a rodent model of volumetric muscle loss injury.</a> Eur Cell Mater. 28, 11-23 (2014).</li><li>Laschke, M. W., 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=Adipose+tissue-derived+microvascular+fragments+from+aged+donors+exhibit+an+impaired+vascularisation+capacity.">Adipose tissue-derived microvascular fragments from aged donors exhibit an impaired vascularisation capacity.</a> Eur Cell Mater. 28, 287-298 (2015).</li><li>Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Green+mice'+as+a+source+of+ubiquitous+green+cells.">'Green mice' as a source of ubiquitous green cells.</a> FEBS Lett. 407 (3), 313-319 (1997).</li><li>Honek, 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=Modulation+of+age-related+insulin+sensitivity+by+VEGF-dependent+vascular+plasticity+in+adipose+tissues.">Modulation of age-related insulin sensitivity by VEGF-dependent vascular plasticity in adipose tissues.</a> Proc Natl Acad Sci U S A. 111 (41), 14906-14911 (2014).</li><li>Cho, C. 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=Angiogenic+role+of+LYVE-1-positive+macrophages+in+adipose+tissue.">Angiogenic role of LYVE-1-positive macrophages in adipose tissue.</a> Circ Res. 100 (4), e47-e57 (2007).</li><li>Han, S., Sun, H. M., Hwang, K. C., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-Derived+Stromal+Vascular+Fraction+Cells:+Update+on+Clinical+Utility+and+Efficacy.">Adipose-Derived Stromal Vascular Fraction Cells: Update on Clinical Utility and Efficacy.</a> Crit Rev Eukaryot Gene Expr. 25 (2), 145-152 (2015).</li><li>Chen, Y. 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Dans cette étude, nous présentons un protocole bien établi pour l&#39;isolement des ad-MVF. L&#39;obtention ad-MVF du tissu adipeux de souris est une procédure simple avec quelques étapes critiques. Les souris présentent différents dépôts de graisse sous-cutanée et intra-abdominales. Comme précédemment décrit pour les rats, la source de matières grasses la plus appropriée pour l&#39;isolement de l&#39; ad-MVF sont les coussinets adipeux épididymaire en raison de leur taille, de structure homogène et une...

L&#39; ingénierie tissulaire se concentre sur la fabrication de produits de remplacement de tissus et d&#39; organes qui maintenir, rétablir ou d&#39; augmenter la fonction des inopérable homologues in vivo 1, 2. Le sort des constructions de l&#39; ingénierie tissulaire dépend essentiellement d&#39; une vascularisation adéquate 3. Réseaux microvasculaires au sein de ces constructions doivent être organisés hiérarchiquement avec artérioles, capillaires et veinules pour permettre la perfusion sanguine efficace après anastomose à la vasculature du bénéficiaire 4. La génération de ces réseaux est parmi les principaux défis dans l&#39;ingénierie tissulaire. A cet effet, un large éventail de stratégies de revascularisation expérimentales a été mis en place au cours des deux dernières décennies , 5, 6. approches angiogéniques stimulent la croissance interne des microvaisseaux bénéficiaires dans Tiss d&#39;ingénierieues par des moyens de modification de structure d&#39;échafaudage ou physico - chimique, telles que l&#39;incorporation de facteurs de croissance 7. Toutefois, pour les grandes constructions de vascularisation en trois dimensions, des stratégies dépendant de l&#39; angiogenèse sont nettement limités par des taux de croissance lente de développement microvaisseaux 8. Au contraire, le concept de prévascularisation vise pour la génération des réseaux microvasculaires fonctionnelles au sein des constructions de tissus avant leur implantation 9. Prévascularisation classique implique la co-culture de cellules de formation de vaisseaux, comme les cellules endothéliales, les cellules murales ou des cellules souches 10, à l&#39; intérieur des échafaudages. Après la formation du réseau microvasculaire, les constructions prévascularisées peuvent alors être implantés dans des défauts du tissu. Il convient de noter, cette approche prévascularisation est difficile à appliquer dans un contexte clinique, car elle est basée sur in vitro complexe et prend du temps &lt;/ em&gt; procédures, qui sont limitées par les principaux obstacles réglementaires 9. Par conséquent, il y a encore un besoin pour le développement de nouvelles stratégies de prévascularisation qui sont plus appropriés pour une large application clinique. Une telle stratégie de prévascularisation peut être l&#39;application des dérivées de tissu adipeux fragments microvasculaires (ad-MVF). ad-MVF représentent des unités de vascularisation puissants qui peuvent être récoltées en grandes quantités dans le tissu adipeux des rats 11, 12 et 13 les souris. Ils se composent d&#39;artérioles, capillaires et veinules segments de vaisseaux, qui présentent une morphologie de microvaisseaux physiologique avec une lumière et de stabilisation des cellules périvasculaires 14, 15. Cette caractéristique unique permet l&#39;implantation immédiate des échafauds ad MVF tête de série dans les défauts des tissus sans préculture. Là-bas, l&#39;annonce-MVF réassembler rapidementà des réseaux microvasculaires fonctionnelles. Par ailleurs, ad-MVF représentent une riche source de cellules souches mésenchymateuses 16, ce qui peut contribuer en outre à leur capacité de régénération frappant. Par conséquent, ad-MVF sont de plus en plus utilisé dans différents domaines de l&#39; ingénierie tissulaire 14, 15, 17, 18, 19, 20, 21. L&#39;isolement de l&#39; ad-MVF a été initialement mis en place chez les rats 11, 12. Nous décrivons ici un protocole qui permet l&#39;isolement normalisé ad-MVF de souris à partir de tampons de graisse épididyme. Cela peut fournir de nouvelles informations sur les mécanismes moléculaires sous-jacents fonction ad-MVF en utilisant des modèles de souris transgéniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

Un réseau microvasculaire fonctionnelle est d&#39;une importance cruciale pour la survie et l&#39;intégration des constructions de tissu d&#39;ingénierie. A cet effet, plusieurs stratégies angiogéniques et prévascularisation ont été mis en place. Cependant, la plupart des approches basées sur les cellules comprennent chronophages étapes in vitro pour la formation d&#39;un réseau microvasculaire. , Ils ne sont donc pas adaptés pour peropératoires procédures en une seule étape. fragments microvasculaires adipeuses dérivées de tissu (ad MVF) représentent des unités de vascularisation prometteuses. Ils peuvent facilement être isolées à partir de tissu adipeux et présentent une morphologie fonctionnelle microvaisseaux. De plus, ils réassembler rapidement dans de nouveaux réseaux microvasculaires après l&#39; implantation in vivo. En outre, ad-MVF ont été montré pour induire lymphangiogenèse. Enfin, ils sont une riche source de cellules souches mésenchymateuses, qui peut en outre contribuer à leur fort potentiel de vascularisation. Dans des études précédentes, nous avons démontré la vascularizati remarquablesur la capacité d&#39;ad-MVF dans l&#39;os d&#39;ingénierie et de substituts cutanés. Dans la présente étude, nous présentons un protocole standardisé pour l&#39;isolement enzymatique ad-MVF de tissu adipeux de souris.

Dans la présente étude, nous avons effectué six procédures d&#39;isolement ad MVF avec le tissu adipeux de 7 à 12 mois vieux mâle de type sauvage C57BL / 6 souris (poids corporel moyen: 35 ± 1 g). La figure 1 illustre la récolte des plaquettes murines de graisse épididymaire avec l&#39; isolation ad-MVF mécanique et enzymatique subséquente. Le temps nécessaire à la récolte de matières grasses était de 30 minutes et pour l&#39;isolement des ad-MVF était d...

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

Nous présentons un protocole pour isoler des fragments microvasculaires du tissu adipeux dérivé qui représentent des unités de vascularisation prometteuses. Ils peuvent être isolés rapidement, ne nécessitent pas dans le traitement in vitro et, par conséquent, peut être utilisé pour prévascularisation une étape dans différents domaines de l&#39; ingénierie tissulaire.

Isolement des murins dérivés tissu adipeux microvasculaire fragments comme Vascularization unités pour l&#39;ingénierie tissulaire

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

The overall goal of this procedure is to isolate microvascular fragments from murine adipose tissue for the generation of vascularization units for tissue engineering. This method can help answer key questions in the field of tissue engineering about the in vivo vascularization of tissue substitues. An advantage of this technique is that it yields fully functional microvascular segments without complex in vitro processing that can be seeded onto scaffolds and implanted into tissue defects.This method can also be applied to other systems such as in vitro angiogenesis assays to unravel molecular mechanisms underlying microvascular network formation and remodeling. Begin by placing an anesthetized male wild type C57 black six mouse in the supine position under a surgical stereomicroscope and confirm a lack of response to toe pinch. Immobilize the paws with tape and disinfect the abdomen.Next, use dissecting scissors to separate the abdominal layer from the underlying muscle tissue and make a midline laparotomy. Laterally unfold the flaps of the abdominal wall and bilaterally identify the testes, epididymus and the epididymal fat pads. Then use small preparation scissors and fine forceps to harvest the fat pads, taking care not to collect any epididymal tissue and place the fat pads into a petri dish containing 15 ml of 37 degrees celsius DMEM.In a laminar flow hood, wash the fat pads in three separate petri dishes containing 15 ml of PBS and place the washed fat into an empty 14 ml polypropylene tube. Use a tube scale to determine the volume of harvested fat. Then use fine scissors to mechanically mince the fat tissue.When a homogenous tissue suspension is obtained, use a 10 mL measuring pipet to transfer the minced tissue with two volumes of collaginase NB 4G into a 50 mL erlenmeyer flask containing a 25 millimeter stir bar. Place the flask in a cell culture incubator for 10 minutes under vigorous stirring. Then, observe 10 mcL of the digested tissue under a microscope.The most difficult step in this isolation procedure is identifying the appropriate time to stop the digestion. Digesting the fat pads for too long will result in a single cell suspension without any microvascular fragments. If the digestate contains free adipose tissue derived microvascular fragments next to single cells, neutralize the enzyme with two volumes of PBS supplemented with 20%FCS and transfer the cell vessel suspension into new polypropylene tubes.Incubate the solution for five minutes at 37 degrees celsius to separate the microvascular fragments from the remaining fat by gravity. Then, use a one mL precision pipet to carefully remove one mL of the main fat supernatent followed by the use of a 100 mcL precision pipet to remove the rest of the supernatent until the suspension appears to be fat free. When all of the fat has been removed, use a 10 mL measuring pipet to filter the cell vessel suspension through a 50 micron strainer over a 50 mL conical tube and transfer the filtered suspension into a new 14 mL polypropylene tube for each individual microvascular fragment isolate.Collect the microvascular fragment pellets by centrifugation and remove all but the last one mL of supernatent from each tube. Resuspend the pellets in the remaining supernatents. Then, transfer each suspension into individual 1.5 mL conical microcentrifuge tubes and pellet the microvascular fragments again for resuspension in the appropriate final volume of PBS and 20%FCS for the planned downstream analysis.Here, the length distribution of freshly isolated microvascular fragments from six, seven to twelve month old wild type C57 black six mice is shown, with a mean average length of approximately 42 micrometers observed. As this representative cell vessel suspension smear demonstrates, large, medium and small microvascular fragments as well as single cells are observed when the fat tissue has been sufficiently digested. Higher magnification reveals that the microvascular fragments exhibit a mature microvessel morphology with hierarchical microvessel segments.Characterization of the microvascular fragments by flow cytometry indicates that they contain CD31 positive endothelial cells, alpha smooth muscle acton positive paravascular cells and CD117 positive mesenchymal stem cells, marker expressing cells. After seeding on a dermal skin substitute, larger microvascular fragments are mainly localized on the implant's surface with some capillary vessel segments detected within the implant cores. Immunohystochemical staining of the seeded microvascular fragments further reveals physiological microvessel configurations with arteriolar, venular and capillary like fragments.Once mastered, this technique can be completed in two hours if performed properly. After watching this video, you should have a good understanding of how to isolate microvascular fragments from murine adipose tissue as vascularization units for tissue engineering.

Research

JoVE Journal

Bioengineering

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Recherche

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Postproduction Processing of Electrospun Fibres for Tissue Engineering

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Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of the Intestine in a Murine Model

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Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

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Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

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Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Génie tissulaire par Intrinsic vascularisation dans un<em&gt; In Vivo</em&gt; Tissue Chambre Ingénierie

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

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Une plate-forme microfluidique pour l&#39;isolement à haut débit unique cellule et Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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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 ...