Name: isolation of murine adipose tissue-derived microvascular fragments as vascularization units for tissue engineering
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אנו מודים על הסיוע הטכני המצוין של ג&#39;נין Becker, קרוליין Bickelmann ורות ניקלס. מחקר זה מומן על ידי מענק של (DFG - קרן המחקר הגרמנית) - LA 2682 / 7-1.

כל הנהלים בוצעו על פי המכון הלאומי הנחיות הבריאות לשימוש בחיות מעבדה ועקבו הנחיות מוסדיים (Landesamt für Soziales, Gesundheit und Verbraucherschutz, אבט. Lebensmittel- und Veterinärwesen, ה&quot;צנטרלשטלה&quot;, זארבריקן, גרמניה). 1. הכנת מכשירים כירורגיים <ol style=";text-align:right;dire...

<ol>
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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. 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במחקר זה אנו מציגים פרוטוקול ומבוסס על הבידוד של אד-MVF. קבלת פרסומות MVF מרקמות שומן בעכברים היא הליך פשוט עם כמה צעדים קריטיים. עכברים להפגין תת עורית שונה ופיקדונות שומן intraabdominal. כפי שתואר לעיל עבור חולדות, מקור שומן המתאים ביותר עבור בידוד של אד-MVF הם רפידות שומן epididyma...

הנדסת רקמות מתמקדת הייצור של תחליפי רקמה ואיבר מקיימים, לשחזר או להגדיל את הפונקציה של שמיש ב עמיתיהם vivo 1, 2. גורלם של מבני רקמות מהונדסים תלוי באופן מכריע על כלי דם הולם 3. רשתות כלי דם בתוך המבנים האלה צריכים להיות מאורגנים באופן היררכי עם arterioles, נימים, ו venules לאפשר טפטוף דם יעיל אחרי inosculation אל כלי הדם של הנמען 4. הדור של רשתות כאלה הוא בין האתגרים המרכזיים בתחום הנדסת רקמות. לשם כך, קשת רחבה של אסטרטגיות כלי דם ניסיוני כבר הציגה במהלך שני העשורים האחרונים 5, 6. גישות אנגיוגנזה לעורר את ingrowth של microvessels הנמען Tiss מהונדסיםUES באמצעות שינוי פיגום מבני או physicochemical, כגון השילוב של צמיחת גורמי 7. עם זאת, עבור כלי דם של המבנים התלת-ממדי גדול, אסטרטגיות תלויי אנגיוגנזה מוגבלים במידה ניכרת בשיעורי צמיחה איטית של פיתוח microvessels 8. לעומת זאת, מושג prevascularization שואף עבור הדור של רשתות כלי דם פונקציונליות בתוך רקמת הבונה לפני ההשתלה שלהם 9. Prevascularization הקונבנציונלי כרוך לתרבות המשותפת של תאים מייצרי כלי, כגון תאי אנדותל, תאי ציור קיר או תאי גזע 10, בתוך פיגומים. לאחר היווצרות רשת כלי דם, מבני prevascularized לאחר מכן ניתן מושתלים לתוך פגמים ברקמות. ראוי לציון, גישת prevascularization זה קשה ליישם במסגרת רפואית, משום שהיא מבוססת על קומפלקס זמן רב במבחנה &lt;/ em&gt; נהלים, אשר מוגבלים על ידי מכשולים רגולטוריים מרכזיים 9. בהתאם לכך, יש עדיין צורך לפיתוח אסטרטגיות prevascularization רומן כי הם יותר מתאימים יישום קליני רחב. אסטרטגיה כזו prevascularization עשויה להיות היישום של שברי כלי דם נגזר רקמה שומני (ad-MVF). אד-MVF מייצג יחידות של כלי דם חזקים כי ניתן לקצור בכמויות גדולות מרקמת השומן של חולדות 11, 12 ו עכברים 13. הם מורכבים arteriolar, נימים, ומגזרי כלי venular, אשר מציגים מורפולוגיה microvessel פיזיולוגית עם לומן ותאי perivascular ייצוב 14, 15. תכונה ייחודית זו מאפשרת ההשתלה המיידית של פיגומי אד-MVF זרע לתוך פגמי רקמות ללא precultivation. יש, במודעה-MVF במהירות להרכיב מחדש בלרשתות כלי דם פונקציונליות. יתר על כן, אד-MVF מייצג מקור עשיר של תאי גזע mesenchymal 16, אשר עשוי גם לתרום יכולת ההתחדשות הבולטת שלהם. בהתאם לכך, אד-MVF משמש יותר ויותר בתחומים שונים של הנדסת רקמות 14, 15, 17, 18, 19, 20, 21. בידודו של אד-MVF כבר נקבע במקור בחולדות 11, 12. בזאת, אנו מתארים פרוטוקול, אשר מאפשר בידוד הסטנדרטי של MVF מודעה בעכברים מן רפידות שומן epididymal. זה עשוי לספק תובנות נוספות המנגנונים המולקולריים שבבסיס פונקציה אד-MVF באמצעות מודלים עכבר מהונדס.

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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

רשת כלי דם פונקציונלית היא בעל חשיבות המרכזית להישרדות והאינטגרציה של מבני רקמות מהונדסים. לשם כך, מספר אסטרטגיות אנגיוגנזה ו prevascularization הוקמו. עם זאת, רוב הגישות מבוססות תאים כוללות גוזלת זמן במבחנת צעדים ליצירת רשת כלי דם. לפיכך, הם אינם מתאימים נהלי צעד אחד תוך ניתוחיים. רקמת שומן הנגזרת שברי כלי דם (ad-MVF) מייצג יחידות כלי דם הבטיח. הם יכולים להיות בקלות לבודד מרקמות שומן ולהציג מורפולוגיה microvessel פונקציונלית. יתר על כן, הם להרכיב מחדש במהירות לתוך רשתות כלי דם חדשות לאחר השתלת in vivo. בנוסף, אד-MVF הוכח לגרום lymphangiogenesis. לבסוף, הם מקור עשיר של תאי גזע mesenchymal, אשר עשוי לתרום לפוטנציאל כלי הדם הגבוה שלהם. במחקרים קודמים אנו הדגמנו את vascularizati המדהיםעל יכולת של MVF מודעת תחליפי עצם ועור מהונדסים. במחקר הנוכחי, אנו מדווחים על פרוטוקול סטנדרטי עבור הבידוד אנזימטי של אד-MVF מרקמות שומן בעכברים.

במחקר הנוכחי ביצענו שש פרוצדורות בידוד אד-MVF עם רקמת השומן בין 7 ל wild-type זכר בן 12 חודש C57BL / 6 עכברים (משקל הגוף הממוצע: 35 ± 1 g). איור 1 מדגים את הקצירה של רפידות שומן epididymal בעכברים עם בידוד מכנים האנזימטית שלאחר מכן אד-MVF. הזמן הדרוש קצירת השומן היה...

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

אנו מציגים פרוטוקול לבודד שברי כלי דם ברקמה שומני נגזרות המייצגים יחידות כלי דם הבטיח. הם יכולים להיות מבודדים במהירות, אינו דורש עיבוד במבחנה, ובכך, ניתן להשתמש prevascularization צעד אחד בתחומים שונים של הנדסת רקמות.

בידוד של נגזרות רקמה שומנית Murine כלי דם שבר כיחידות כלי דם של הנדסת רקמות

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

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

PGS אלסטומרי פיגומים להנדסת רקמות העורקים

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Postproduction Processing of Electrospun Fibres for Tissue Engineering

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רקמות שריר שלד הנדסה מתאי Murine היצמדות מולידו ויישום של גירוי חשמלי

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

הנדסת רקמות של המעי בדגם Murine

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Capillary Force Lithography for Cardiac Tissue Engineering

נימי חיל יתוגרפיה להנדסת רקמות לב

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

הנדסת ג&#39;ל קולגן Cellularized 3D לשחזור רקמות וכלי דם

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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 ...

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

הנדסת רקמות ידי עצמותי כלי דם בתוך<em&gt; In vivo</em&gt; הנדסת רקמות קאמרית

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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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 ...