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

We are grateful for the excellent technical assistance of Janine Becker, Caroline Bickelmann and Ruth Nickels. This study was funded by a grant of the Deutsche Forschungsgemeinschaft (DFG - German Research Foundation) - LA 2682/7-1.

All procedures were performed according to the National Institute of Health guidelines for the use of experimental animals and followed institutional guidelines (Landesamt f&#252;r Soziales, Gesundheit und Verbraucherschutz, Abt. Lebensmittel- und Veterin&#228;rwesen, Zentralstelle, Saarbr&#252;cken, Germany).
1. Preparation of Surgical Instruments
<ol>
	<li>Keep ready the dissection scissors, surgical forceps, small preparation scissors, fine forceps and a sterile Petri dish with 15 mL Dulb...

<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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+in+tissue+engineering:+angiogenesis+versus+inosculation.">Vascularization in tissue engineering: angiogenesis versus inosculation.</a> Eur Surg Res. 48 (2), 85-92 (2012).</li><li>Sarker, M., Chen, X. B., Schreyer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+approaches+to+vascularisation+within+tissue+engineering+constructs.">Experimental approaches to vascularisation within tissue engineering constructs.</a> J Biomater Sci Polym Ed. 26 (12), 683-734 (2015).</li><li>Frueh, F. S., Menger, M. D., Lindenblatt, N., Giovanoli, P., Laschke, M. 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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. 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In this study we present a well-established protocol for the isolation of ad-MVF. Obtaining ad-MVF from murine adipose tissue is a straightforward procedure with a few critical steps. Mice exhibit different subcutaneous and intraabdominal fat deposits. As previously described for rats, the most suitable fat source for the isolation of ad-MVF are the epididymal fat pads due to their size, homogeneous structure and minimal contamination with larger blood vessels11,12</su...

Tissue engineering focuses on the fabrication of tissue and organ substitutes that maintain, restore or augment the function of inoperable in vivo counterparts1,2. The fate of engineered tissue constructs crucially depends on an adequate vascularization3. Microvascular networks within these constructs should be hierarchically organized with arterioles, capillaries, and venules to allow efficient blood perfusion after inosculation to the recipient&#39;s vasculature4. The generation of such networks is among the key challenges in tissue engineering. For this purpose, a broad spectrum of experimental vascularization strategies has been introduced over the last two decades5,6.
Angiogenic approaches stimulate the ingrowth of recipient microvessels into engineered tissues by means of structural or physicochemical scaffold modification, such as the incorporation of growth factors7. However, for the vascularization of large three-dimensional constructs, angiogenesis-dependent strategies are markedly limited by slow growth rates of developing microvessels8.
In contrast, the concept of prevascularization aims for the generation of functional microvascular networks within tissue constructs prior to their implantation9. Conventional prevascularization involves the co-culture of vessel-forming cells, such as endothelial cells, mural cells or stem cells10, within scaffolds. After microvascular network formation, the prevascularized constructs can then be implanted into tissue defects. Noteworthy, this prevascularization approach is difficult to apply in a clinical setting, because it is based on complex and time-consuming in vitro procedures, which are restricted by major regulatory hurdles9. Accordingly, there is still a need for the development of novel prevascularization strategies that are more suitable for a broad clinical application.
Such a prevascularization strategy may be the application of adipose tissue-derived microvascular fragments (ad-MVF). ad-MVF represent potent vascularization units that can be harvested in large amounts from the fat tissue of rats11,12 and mice13. They consist of arteriolar, capillary, and venular vessel segments, which exhibit a physiological microvessel morphology with a lumen and stabilizing perivascular cells14,15. This unique feature allows the immediate implantation of ad-MVF-seeded scaffolds into tissue defects without precultivation. There, the ad-MVF rapidly reassemble into functional microvascular networks. Furthermore, ad-MVF represent a rich source of mesenchymal stem cells16, which may additionally contribute to their striking regenerative capacity. Accordingly, ad-MVF are increasingly used in different fields of tissue engineering14,15,17,18,19,20,21.
The isolation of ad-MVF has originally been established in rats11,12. Herein, we describe a protocol, which allows the standardized isolation of murine ad-MVF from epididymal fat pads. This may provide further insights into molecular mechanisms underlying ad-MVF function by using transgenic mouse models.

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

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

In the present study we performed six ad-MVF isolation procedures with fat tissue from 7- to 12-month-old male wild-type C57BL/6 mice (mean body weight: 35 &#177; 1 g). Figure 1 illustrates the harvesting of murine epididymal fat pads with subsequent mechanical and enzymatic ad-MVF isolation. The time required for the harvesting of fat was 30 min and for the isolation of ad-MVF was 120 min. In total, the procedure took 150 min.
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Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

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.

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