Name: creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model
Uploaded: 2017-08-04T12:30:00
Description: Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

Forfatterne takker Dr. Yukiko Koga avdeling av plast og rekonstruktiv kirurgi, Juntendo University School of Medicine, for å gi praktiske råd. Vi har også takke Mr. Hidekazu Murata av diabetiker sentrum av Tokyo kvinnenes Medical University School of Medicine utmerket kundestøtte. Denne studien ble støttet av etableringen av innovasjon sentrene for avanserte tverrfaglig forskning områder programmet prosjektets for utvikling innovasjon systemer "celle ark Tissue Engineering Center (CSTEC)" fra Kunnskapsdepartementet, kultur, sport, vitenskap og teknologi (MEXT) i Japan.

All experimental protocols presented below were approved by the Animal Welfare Committee of Tokyo Women&#39;s Medical University School of Medicine and abided by all requirements of the Guidelines for Proper Conduct of Animal Experiments.
1. Preparation of Animals, Instruments, Culture Media, and Dishes
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	<li>Prepare complete culture medium using minimum essential medium alpha containing 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Store this for several months at 4 &#176...

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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=Wound+healing+effect+of+adipose-derived+stem+cells:+a+critical+role+of+secretory+factors+on+human+dermal+fibroblasts.">Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts.</a> J Dermatol Sci. 48 (1), 15-24 (2007).</li><li>Nakagami, 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=Novel+autologous+cell+therapy+in+ischemic+limb+disease+through+growth+factor+secretion+by+cultured+adipose+tissue-derived+stromal+cells.">Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells.</a> Arterioscler Thromb Vasc Biol. 25 (12), 2542-2547 (2005).</li><li>Asahara, T., 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=VEGF+contributes+to+postnatal+neovascularization+by+mobilizing+bone+marrow-derived+endothelial+progenitor+cells.">VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.</a> EMBO J. 18 (14), 3964-3972 (1999).</li><li>Kato, Y., 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=Allogeneic+transplantation+of+an+adipose-derived+stem+cell+(ASC)+sheet+combined+with+artificial+skin+accelerates+wound+healing+in+a+rat+wound+model+of+type+2+diabetes+and+obesity.">Allogeneic transplantation of an adipose-derived stem cell (ASC) sheet combined with artificial skin accelerates wound healing in a rat wound model of type 2 diabetes and obesity.</a> Diabetes. , db141133 (2015).</li></ol>

De viktigste trinnene for dyrking er en rASC ark er som følger: 1) temperaturen må opprettholdes på ca 37 ° C under dyrking på temperatur-responsive kultur retter. Under opprettingen av en rASC ark, hver prosedyren ble utført på en 37 ° C thermo-plate, og hver reagens ble varmet til 37 ° C for å hindre at cellene spontant frakobling fra parabolen31. 2) mottaker ZDF rotter må overvåkes for å hindre fjerning av ikke-klebende dressing, som er avgjørende for vellykket transplantasjon av ...

Befolkningen i diabetespasienter er økende over hele verden og nådde 400 millioner i 20151; rundt 15-25% av pasienter med diabetes er utsatt for utviklingen av en nedre ekstremitet diabetiker sår2. Nedre ekstremitet diabetiker sår er vanskelige og krever en terapeutisk lengre med rehabilitering trening etter fullstendig gjenoppretting. En lang behandling periode ofte resulterer i en betydelig reduksjon i pasienten livskvalitet. Derfor må det utvikles nye behandlingsformer som redusere eller hindre forverring for behandling av diabetiker sår. For å evaluere diabetiker sårheling, vi optimalisert en diabetiker sår sår-tilheling modell i rotter, som etterligner praktisk klinisk betingelser, og vurdert om transplanting liggende under adipose-avledet stilk cellen (ASC) ark ved hjelp av cellen arks engineering akselerert sårtilheling.
Mesenchymal stromal celler (MSCs) viser en utmerket potensial for akselererende sårheling på grunn av deres selvtillit fornyelse kapasitet, deres immunmodulerende effekter og evnen til å differensiere i ulike celle linjene3. ASCs er en type MSC Hentet fra fettvev, og de viser flere fordeler sammenlignet med MSCs avledet fra andre vev, inkludert angiogenic potensielle og paracrine aktivitet4,5. Fettvev er relativt rikt på kroppen sin tilgjengelighet tillater samlingen med minimal invasiv prosedyrer. Derfor har ASCs blitt brukt eksperimentelt for sårhelende programmer6,7.
Tidligere rapporter har vist at den direkte injeksjon av encellede MSC suspensjoner i områdene rundt sår kan akselerere sårheling8,9. Men til tross for rapporter om akselerasjon av sårheling diabetiker sår modeller etter injeksjon av encellede suspensjoner, er overlevelse tidspunktet for transplantert celler i såret området ikke klart.
I denne studien brukt vi celle arks engineering bruker temperatur-responsive kultur retter. Disse rettene har temperaturen svarer polymer N- isopropylacrylamide covalently bundet til deres overflate10. Podet polymer laget tillater temperaturkontrollert celle vedheft til eller avdeling fra overflaten av kultur parabolen. Overflaten av fatet blir hydrofobe ved 37 ° C, slik at cellene å overholde og sprer, mens celler spontant koble fra overflaten når det blir hydrofile ved temperaturer under 32 ° C. Kultivert celler kan høstes som sammenhengende celle ark med intakt celle til celle veikryss og ekstracellulære matriser (ECMs) ved å redusere temperaturen; Dermed er proteolytiske enzymer som skade ECM, som trypsin, ikke nødvendig11. Derfor kan celle arks engineering bevare celle til celle tilkoblinger og forbedre effekten av cellen transplantasjon.
I tillegg øker celle arks transplantasjon celle overlevelse sammenlignet med celle injeksjon12. I denne protokollen, ble Zucker diabetiker fet (ZDF) rotter valgt som type 2 diabetes og fedme modell med forsinket sårtilheling. ZDF rotter utvikle spontant fedme på ca 4 uker. De deretter utvikle type 2 diabetes med fedme mellom 8 og 12 ukens av alderen, da de viser hyperglykemi assosiert med insulin resistens, dyslipidemi og hypertriglyceridemia13. Forsinket sårheling, redusert blodstrøm i perifere blodkar og diabetiker nephropathy er også observert14,15,16. Videre kan ZDF rotter være en passende modell for å studere helbredelse av uløselige kutan sår, som diabetisk magesår.
Forskjellene mellom mennesker og gnagere i sårhelende mekanismer er forbundet med anatomiske forskjeller i huden. Sårheling i vanlig rotter er basert på såret kontraksjon, mens sårheling hos mennesker er basert på nytt epithelialization og korning vev formasjonen. Vanligvis såret splinting brukes på gnager modeller bidrar til å minimere såret sammentrekning og gir gradvis dannelsen av korning vev17, selv om sår i nondiabetic rotter er nesten helt lukket av sammentrekning. Imidlertid diabetiker sår sammentrekning i ZDF rotter er svekket, og sårtilheling primært oppstår gjennom re epithelialization og korning vev formasjonen; Dermed er denne prosessen mer lik menneskelige sårheling14.
Diabetiker sår med utsatt bein etter rensing er ofte møtte klinisk. Tidligere studier har undersøkt 12 mm diameter full-tykkelse huden sår på ryggen av athymic naken mus18,19 og 10 mm diameter full-tykkelse huden sår på ryggen av normal mus20. For å utvikle en klinisk modell for alvorlig diabetiker sår, større (15 x 10 mm2) full-tykkelse hud defekter med synlige og uten periosteum ble opprettet, som beskrevet tidligere21, i rotter med type 2 diabetes og fedme.
Rotte ASC (rASC) ark fra ASCs normal Lewis rotter ble opprettet via allogenic transplantasjon av ASC ark. I klinisk praksis er autolog transplantasjon drivverdige fordi diabetiker pasienter med sår oppfører seg ofte alvorlig diabetiker komplikasjoner, som ukontrollert høyt blodsukker og høy body mass indekser, og disse komplikasjoner årsaken sårhelende lidelser som øker vanskelighetene med å skaffe fettvev fra disse pasientene. Videre ASCs fra dyr med diabetes utstillingen endret egenskaper og nedsatt funksjon22. Derfor beskriver protokollen presenteres her allogenic transplantasjon av rASC ark fra vanlig rotter og bruk av kunstig huden til diabetiker rotter.
Bilayer kunstig huden brukes i denne protokollen hindrer spontan sammentrekning av sår, fremmer syntesen av en ny bindevev matrise og ligner ekte dermis23. I denne protokollen, er kunstige huden plassert på en rASC ark og fast med nylontråder å forhindre sår sammentrekning eller utvidelse som følge av løs rotte huden. Dessuten, kunstige huden gir en tredimensjonal ramme for ASC arkene opprettholder et fuktig miljø for transplantert ASC ark og sår og beskytter sår fra infeksjon og ytre krefter. Til slutt, en ingen-klebe-dressing er plassert over såret å beskytte den mot eksterne påkjenninger, opprettholde et fuktig såret miljø og absorbere sårvæske.
En rASC er tynn, fleksibel og deformerbare og kan bli overholdt flytte mottaker områder, for eksempel en bankende hjerte24. Celle-ark er brukt for gjenoppbyggingen av ulike vev og kan generere helbredende effekter25,26. ASC ark som stiller ut kliniske terapeutiske potensialet kan akselerere healing for mange typer av sår. Videre allogene transplantasjon av ASC ark, kombinert med bruk av kunstig hud, kanskje er relevante for behandling av vanskelige sår eller forbrenninger, som observert i perifere arterial sykdom eller kollagen sykdom, eller de kan gis til pasienter som er underernærte eller bruker steroider. Denne metoden øker effektiviteten av transplanting ASCs. Sårhelende ZDF rotte modell produserer en alvorlig såret tilstand som ligner menneskelige såret helbredelsesprosessen og etterligner klinisk betingelser i en lite eksperimentelle dyr.

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			<td height="21" style="height:21px;width:223px;">&#945;-MEM glutamax</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:119px;">32571-036</td>
			<td style="width:167px;">Carlsbad, CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Japan Bioserum Co Ltd.</td>
			<td style="width:119px;">S1650-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">10 103 578 001</td>
			<td>Mannheim, Germany</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">60-cm2 Primaria tissue culture dish</td>
			<td>BD Biosciences</td>
			<td>353803</td>
			<td>Franklin Lakes, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate Buffer Saline (PBS)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">1490-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-ascorbic acid phosphate magnesium salt n-hydrate</td>
			<td style="width:169px;">Wako</td>
			<td>013-19641</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">35-mm temperature-responsive culture dish (UpcellTM)</td>
			<td>CellSeed</td>
			<td>NUNC-174904</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microwarm plate (MP-1000)</td>
			<td style="width:169px;">Kitazato Science Co., Ltd.</td>
			<td style="width:119px;">1111</td>
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			<td height="21" style="height:21px;">Rodent mechanical ventilator</td>
			<td>Stoelting</td>
			<td>#50206</td>
			<td>Wood Dale, IL</td>
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			<td height="21" style="height:21px;">4% isoflurane</td>
			<td>Pfizer Japan</td>
			<td>114-13340-3</td>
			<td>Tokyo, Japan</td>
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			<td height="25" style="height:25px;">Artificial skin (Pelnac&#174;)</td>
			<td>Smith &#38; Nephew</td>
			<td>PN-R40060</td>
			<td>&#160;Tokyo, Japan</td>
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			<td>Smith &#38; Nephew</td>
			<td align="right">66800679</td>
			<td>Known as Allevyn non-adhessing&#174; in the United State</td>
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			<td height="42" style="height:42px;">5-0 nylon suture</td>
			<td>Alfresa</td>
			<td style="width:119px;">EP1105NB45-KF2</td>
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			<td height="21" style="height:21px;">20 CELLSTAR TUBES</td>
			<td>greiner bio-one</td>
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			<td height="21" style="height:21px;width:223px;">15mL Centrifuge Tube</td>
			<td>Corning Incorporated</td>
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			<td height="42" style="height:42px;width:223px;">14 GOLDMAN-FOX PERIOSTEAL</td>
			<td>Hu-Friedy</td>
			<td style="width:119px;">P14</td>
			<td>Chicago, IL</td>
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creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model

Kunstig huden har oppnådd betydelig terapeutiske resultater i klinisk praksis. Men kan kunstig hudbehandlinger for sår i diabetespasienter hindret blodstrøm eller store sår bli forlenget. Cellen-basert behandling har dukket opp som en ny teknikk for behandling av diabetiker magesår, og cellen arks engineering har forbedret effekten av cellen transplantasjon. En rekke rapporter har antydet at liggende under adipose-avledet stilk celler (ASCs), en type mesenchymal stromal cell (MSC), ha terapeutiske potensial på grunn av deres relative overflod i fettvev og deres tilgjengelighet for samling i forhold til MSCs fra andre vev. Derfor synes ASCs å være en god kilde til stamceller for terapeutisk bruk. I denne studien ble ASC ark fra epididymal adipose fett av vanlig Lewis rotter opprettet med temperatur-responsive kultur retter og normal kultur medium som inneholder askorbinsyre. ASC arkene ble transplantert inn Zucker diabetiker fatty (ZDF) rotter, en rotte modell av type 2 diabetes og fedme, som viser redusert sårtilheling. Et sår ble opprettet på bakre skallen overflate, ASC ark ble transplantert i såret og en bilayer kunstig hud ble brukt til å dekke ark. ZDF rotter mottatt ASC ark hadde bedre sårtilheling enn ZDF rotter uten transplantasjon av ASC ark. Denne tilnærmingen var begrenset fordi ASC ark er følsomme for tørre forhold, som krever vedlikehold av et fuktig såret miljø. Derfor ble kunstig huden brukt til å dekke ASC arket for å hindre tørking. Allogenic transplantasjon av ASC ark i kombinasjon med kunstig huden kan også gjelde andre vanskelige sår eller forbrenninger, som observert med perifere arterial sykdom og kollagen sykdom, og kan gis til pasienter som er underernærte eller bruker steroider. Denne behandlingen kan dermed være første skritt mot å forbedre behandlingsalternativer for diabetiker sår helbredelse.

Denne protokollen forsøkte å etablere en ny cellen-basert terapi for intractable diabetiker sår. Kort (som illustrert i figur 1), fører allogene rASC ark ble opprettet fra vanlig rotter bruke celle arks engineering og ble deretter transplanted bruker en bilayer av kunstig huden på en full-tykkelse huden feil på rotte diabetiker. Lys mikroskop bilder av et godt eksempel på en rASC ark (figur 2A) og et dårlig eksempel for en rASC ark (figur ...

Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Liggende under adipose-avledet stilk celler (ASCs) er enkelt isolert og høstet fra fettet til vanlig rotter. ASC ark kan lages med cellen arks engineering og kan være transplantert inn Zucker diabetiker fet rotter viser full-tykkelse hud defekter utsatt bein og deretter dekket med en bilayer av kunstig huden.

Opprettelse og transplantasjon av en liggende under Adipose-avledet stilk cellen (ASC) ark i en diabetiker sårhelende modell

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

The overall goal of this procedure is to verify the effect of adipose-derived stem cell sheets combined with artificial skin on wound healing for a large full-thickness skin defect in diabetic rat. This method can help answer key questions about healing of chronic wound, such as diabetic large wound with exposed bone. The main advantage of this technique is that cells are transplanted to the wound in solid sheets using cell sheet technology, and the result is a viable, stable transplant.For a fat pad donor, plan to use eight to 33 week old Lewis rats. At the surgical site, have ready several sterile cotton-tipped applicators, gauzes, a scalpel, a needle holder, operating scissors, a pair of forceps, and 5-0 nylon suture. For temporary tissue placement, load a 100 milliliter petri dish with 10 milliliters of PBS.Weigh the PBS-loaded dish to later find the mass of the harvested tissue. After anesthetizing the rat, position it supine on a sterile drape. Then, shave the operative area and scrub the exposed skin using several alternating scrubs of 70%ethanol.Now, make a three centimeter skin incision in the lateral lower abdomen. Next, expose and dissect the external oblique muscle. Then, expose the epididymal adipose tissue surrounding the epididymis.Next, carefully excise the epididymal adipose tissue. Avoid damaging the epididymis, testes, and epididymal blood vessels. Transfer the tissue into the dish of PBS.Then, close up the donor rat and follow standard postoperative care practices. Isolate the ASCs from two to three grams of harvested adipose tissue. Digest the tissue for an hour in 0.1%type A collagenase to obtain the stromal vascular fraction, or SVF.After purifying the SVF, suspend the SVF in 20 milliliters of media. Split the suspension into two 60 square centimeter culture dishes, and culture them for 24 hours under standard conditions. The next day, wash the plates with PBS three times to remove any unattached cells and debris.After the final wash, add back fresh complete medium. Then, continue to passage the cells until they are nearly sub confluent on the third day of culturing. Then, using one milliliter of 0.25%trypsin EDTA in a brief incubation.Remove the cells from the plate. Do not let the cells be exposed to active trypsin more than 10 minutes. After observing and counting the cells, subculture them at a density of 1, 700 per square centimeter.Then, pass the cells every three days, and complete three passages before proceeding. After passing the subcultured rat ASCs for the third time, plate 150, 000 cells onto a 35 millimeter diameter temperature-responsive culture dish, and two milliliters of culture medium. Culture the dish for three days in an incubator to make a uniform cell sheet.Three days later, change the medium, and add 16.4 micrograms per milliliter of AA.Then culture the cells, changing the AA medium every other day. After four or five days on the AA-containing medium, check the proliferation of the ASCs. Look for gaps between the cells and the contiguous cell sheet.Once a contiguous cell sheet is established, the cells can be used for the model. When needed, collect the cell sheet by placing the plate at room temperature for 15 to 20 minutes. Because the plate is temperature sensitive, the cells will spontaneously detach as a contiguous sheet, and the sheet can be removed with forceps.For the obesity wound healing model, use adult male ZDF rats, 16 to 18 weeks old weighing between 500 and 600 grams. Position and anesthetize rat on the surgical field, and before shaving the head, clean it using 70%ethanol. Then, shave the operative area and clean the exposed skin with alternating scrubs of 70%ethanol.Around this time, transfer the temperature sensitive plate of adipose stem cells to the bench. Before the cell sheet detaches from the plate, remove the medium and wash the cell sheet with PBS three times. After collecting the cell sheet, soak it in PBS until needed.Once the cells are ready, make a 15 by 10 millimeter full-thickness skin defect on the rat's head. First, remove the cutaneous tissue down to the periosteum. Then, excise the skin an cutaneous tissue with a scalpel and a pair of surgical scissors.To complete the defect, remove the periosteum with the periosteal raspatory. Then place the cell sheet over the defect. The stem cell sheet is soft and flexible.Use forceps to extend it to every corner of the defect. Now, cover the cell sheet and defect with a 10 by 15 millimeter patch of artificial skin soaked in saline right before use, and close the wound with approximately 10 stitches using 5-0 nylon suture. To protect the wound, use 5-0 nylon suture to secure a 15 by 20 millimeter patch of non-adhesive dressing over the artificial skin.Plan to observe the dressing every other day, because the rat will invariably remove it, and if needed, replace the dressing promptly. In two groups of six Zucker diabetic fatty rats, the described protocol was used to create a skull defect. Experimental rats were treated with a rat adipose stem cell sheet, and the controls were not.After 14 days, the wounds of the treatment group were significantly smaller than those of the controls. After watching this video, you should have a good understanding of how to make large wound with exposed bone in rats, and then treat that wound with adipose derived stem cells. While attempting this procedure, be sure to use strict temperature management during the entire process of using temperature responsive culture dishes.Otherwise, cells could spontaneously detach from the dishes, delaying your experiments.

Research

JoVE Journal

Medicine

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

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