Name: transplantation of adipose tissue-derived stem cell sheet to reduce leakage after partial colectomy in a rat model
Uploaded: 2018-08-11T12:30:00
Description: Watch this Scientific Journal Video about Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model at JoVE.com

Os autores agradecem ao Prof. Dr. S.E.R. Hovius, Dr. M.A.M. Mureau e todos os cirurgiões do departamento de cirurgia plástica para a coleção de tecido adiposo subcutâneo. P. Sukho é suportado por uma concessão do programa The Netherlands Fellowship (NFP-12/435), durante a realização do estudo. Y.M. Bastiaansen-Jenniskens é suportado por doações da Fundação holandesa de artrite (LP11).

Tecido adiposo abdominal subcutâneo foi obtido de doadores humanos com aprovação do Comité de ética médica (#MEC-2014-092), Erasmus MC University Medical Center, Rotterdam, Holanda. Todos os experimentos com animais foram aprovados pelo Comitê ético da experimentação Animal, Erasmus MC University Medical Center, Rotterdam, Países Baixos (133-14-01).
1. cultura e isolamento de ASCs humana
<ol><li>Prepare-se 3 mL de meio de isolamento para 1 g de tecido adiposo. Mix baixa glicose Du...

<ol>
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Cell Harvesting by Temperature Reduction Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/brochures/AppNote-Nunc-UpCell-N20230.pdf">https://tools.thermofisher.com/content/sfs/brochures/AppNote-Nunc-UpCell-N20230.pdf</a> (2008)</li><li>Wu, Z., 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=Colorectal+anastomotic+leakage+caused+by+insufficient+suturing+after+partial+colectomy:+a+new+experimental+model.">Colorectal anastomotic leakage caused by insufficient suturing after partial colectomy: a new experimental model.</a> Surg Infect (Larchmt). 15 (6), 733-738 (2014).</li><li>Wu, Z., 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=Reducing+colorectal+anastomotic+leakage+with+tissue+adhesive+in+experimental+inflammatory+bowel+disease.">Reducing colorectal anastomotic leakage with tissue adhesive in experimental inflammatory bowel disease.</a> Inflamm Bowel Dis. 21 (5), 1038-1046 (2015).</li><li>Wu, Z., 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=Reducing+anastomotic+leakage+by+reinforcement+of+colorectal+anastomosis+with+cyanoacrylate+glue.">Reducing anastomotic leakage by reinforcement of colorectal anastomosis with cyanoacrylate glue.</a> Eur Surg Res. 50 (3-4), 255-261 (2013).</li><li>Aysan, E., Dincel, O., Bektas, H., Alkan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polypropylene+mesh+covered+colonic+anastomosis.+Results+of+a+new+anastomosis+technique.">Polypropylene mesh covered colonic anastomosis. 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Escapamento anastomótica é o efeito adverso mais grave após a ressecção do cólon com uma anastomose primária. Técnicas ideais para impedir fugas e ruptura anastomótica ainda estão faltando. Aplicação local de uma matriz de biomateriais tem sido conduzida, com resultados variados25,26,27. O objetivo das terapias com células é facilitar o reparo do tecido por substituição de tecido ou a estimulação da cicatrizaç...

Colectomia parcial com uma anastomose primária é comumente realizada cirurgia que pode ser feita para muitas doenças que afetam o cólon como câncer colorretal, doença de Crohn e diverticulite1,2. A mais temida complicação após anastomose colorretal é fuga anastomótica3. Embora tenham sido identificados vários factores de risco associados a vazamentos anastomótica, soluções para a prevenção de vazamento permanecem unkown4,5.
Tecido adiposo-derivado de células do estroma (ASCs) estão associadas com propriedades anti-inflamatórias e trófico6,7, que faz com que estas células prometendo candidatos para terapias regenerativas8. A eficácia de ASCs para promover a cicatrização tecidual foi mostrada em vários tecidos como o músculo cardíaco, pele e esôfago9,10,11,12,13. No entanto, existem poucos relatos sobre o uso de ASCs para promover a cicatrização intestinal. Transplante de local de ASCs para anastomoses Colorretais experimentais através de biosutures ASC-revestido ou serosas injeções de ASCs não mostraram nenhuma melhora ou na cura de14 ou não impediu a fuga anastomótica apesar de um mais favorável anastomótica cura de15.
Transplante de local de ASCs em suspensão ou combinado com biomateriais podem ser associados com retenção insuficiente ou uma distribuição inadequada de células transplantadas11. Célula folha tecnologia16,17 oferece um método inovador de ASC entrega18,19. Portanto, em um estudo anterior, uma nova abordagem foi proposta na quais ASCs organizados em uma célula de folha poderá ser aplicada na anastomose colorretal experimental20. Este estudo demonstrou que o transplante de folha ASC é bem sucedido em reduzir o vazamento de anastomose colorretal após colectomia parcial em um modelo do rato. Este artigo relata a elaboração de folha ASC e técnica cirúrgica de transplante.

<table border="0" cellpadding="0" cellspacing="0" width="689">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">LG DMEM</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">22320022</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Collagenase type I</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">17100-01</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">A9418</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal bovine serum</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">10270-106</td>
			<td style="width:167px;">FBS, ASC isolation and culture</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">3% acetic acid with methylene blue</td>
			<td style="width:115px;">Stemcell Technologies</td>
			<td style="width:191px;">7060</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Gentamicin</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">15750-037</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ampothericin B&#160;</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">15290-018</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Shaker (Gallenkamp Environmental Shaker Model 10X 400)</td>
			<td style="width:115px;">Akribis Scientific</td>
			<td style="width:191px;">F240</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge&#160;</td>
			<td style="width:115px;">Hettichlab&#160;</td>
			<td style="width:191px;">Rotina380</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:216px;">Phosphate buffer saline&#160;</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">14190-094</td>
			<td style="width:167px;">PBS, ASC isolation and culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ascorbic acid-2-phosphate</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">A8960</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">Human recombinant fibroblast growth factor 2&#160;</td>
			<td style="width:115px;">AbD Serotec</td>
			<td style="width:191px;">AF-100-15</td>
			<td style="width:167px;">FGF2, ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin EDTA</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">25200-056</td>
			<td style="width:167px;">ASC culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">D2650-5x</td>
			<td style="width:167px;">DMSO, ASC freezing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Thermo-responsive culture dishes</td>
			<td style="width:115px;">Nunc Thermo scientific</td>
			<td style="width:191px;">174904</td>
			<td style="width:167px;">ASC sheet preperation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Non-absorbable braided silk 4/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">21151042</td>
			<td style="width:167px;">surgery</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Non-absorbable monofilament polyamide&#160; 8/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">G1118170</td>
			<td style="width:167px;">surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Absorbable braided polyglycolic acid 5/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">C1048207</td>
			<td style="width:167px;">surgery</td>
		</tr>
	</tbody>
</table>

transplantation of adipose tissue-derived stem cell sheet to reduce leakage after partial colectomy in a rat model

Anastomótica escapamento é uma complicação catastrófica após cirurgia colorectal. Embora os métodos atuais para prevenção de vazamento tem diferentes níveis de eficácia clínica, eles são até agora imperfeitas soluções. Terapia de células-tronco, usando folhas de ASC poderia fornecer uma solução para este problema. ASCs são considerados candidatos mais promissores para a promoção do tecido de cicatrização devido às suas propriedades tróficas e imunomoduladores. Aqui, nós fornecemos métodos para produzir folhas ASC de alta densidade, que são transplantadas para uma anastomose colorretal em um modelo do rato para reduzir o vazamento. ASCs formaram folhas de célula em pratos de cultura thermo-responsivo que poderiam facilmente ser desanexados. No dia do transplante, foi realizada uma colectomia parcial com uma anastomose colorretal 5-sutura. Os animais foram transplantados imediatamente com 1 folha ASC por rato. Folhas ASC aderiram espontaneamente para a anastomose sem qualquer cola, sutura ou qualquer biomaterial. Grupos de animais foram sacrificados 3 a 7 dias no pós-operatório. Em comparação com animais transplantados, a incidência de abscessos anastomótica e escapamento foi maior nos animais controle. Em nosso modelo, o transplante de folhas ASC após anastomose colorretal foi bem sucedida e associado uma menor taxa de fugas.

Um fluxograma deste estudo descrevendo tanto cultura folha ASC e o procedimento de ressecção do cólon e anastomose é mostrado na Figura 1. A Figura 2 mostra a morfologia microscópica de folha ASC e a aparência macroscópica da folha ASC durante e após o desprendimento. A Figura 3 mostra os diferentes passos do destacamento de folha ASC e transplante de órgãos. A Figura 4...

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Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Transplante de células-tronco derivadas de tecido adiposo folha para reduzir o vazamento após colectomia parcial em um modelo do rato

A preparação e a transplantação de lençóis de células-tronco (ASC) derivado de tecido adiposo para anastomoses Colorretais insuficientemente suturados em um modelo do rato é apresentado. Esta aplicação romance mostra que folhas ASC podem reduzir o vazamento de anastomose colorretal.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Transplante ortotópico de aorta: um modelo de rato para estudar o desenvolvimento de vasculopatia crônica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Medicina

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

Um modelo do rato de No Utero Transplante

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Transplante de Células-tronco em um In vitro Modelo de isquemia / reperfusão Simulated

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Procedimentos cirúrgicos para um modelo de rato de Transplante de Fígado parcial ortotópico com reconstrução arterial hepática

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Neural Stem Cell Transplantation em Experimental contusão Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

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

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

Criação e transplante de uma folha de células-tronco adiposo-derivado (ASC) em um modelo de cicatrização de feridas diabética

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

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

ADSC folhas transplante para prevenir a estenose após estendido esofágico endoscópica submucosa Dissection

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Intrasplenic transplante de hepatócitos após Hepatectomy parcial na NOD. Ratos SCID

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Re-Arterialized transplante de fígado parcial de rato com na vivo orientado a embarcação 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

Complicações reduzidas após reconexão arterial em um modelo de rato de transplante de fígado ortotópico

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...