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

The authors are grateful to Prof. Dr. S.E.R. Hovius, Dr. M.A.M. Mureau and all surgeons of the department of Plastic Surgery for the collection of subcutaneous adipose tissue. P. Sukho is supported by a grant from The Netherlands Fellowship program (NFP-12/435), during the conduct of the study. Y.M. Bastiaansen-Jenniskens is supported by grants from Dutch Arthritis Foundation (LP11).

Subcutaneous abdominal adipose tissue was obtained from human donors with approval of the Medical Ethical Committee (#MEC-2014-092), Erasmus MC University Medical Center, Rotterdam, The Netherlands. All animal experiments were approved by the Ethical Committee of Animal Experimentation, Erasmus MC University Medical Center, Rotterdam, The Netherlands (133-14-01).
1. Human ASCs Isolation and Culture
<ol>
	<li>Prepare 3 mL of isolation medium for 1 g of adipose tissue. Mix low glucose Dulbecco...

<ol>
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F. <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:+aspects+of+prevention,+detection+and+treatment.">Colorectal anastomotic leakage: aspects of prevention, detection and treatment.</a> World J Gastroentero. 19 (15), 2293-2297 (2013).</li><li>Testini, 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=Bovine+pericardium+patch+wrapping+intestinal+anastomosis+improves+healing+process+and+prevents+leakage+in+a+pig+model.">Bovine pericardium patch wrapping intestinal anastomosis improves healing process and prevents leakage in a pig model.</a> PLoS One. 9 (1), e86627 (2014).</li><li>Kilroy, G. 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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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Anastomotic leakage is the most serious adverse event following colon resection with a primary anastomosis. Optimal techniques to prevent anastomotic disruption and leakage are still lacking. Local application of an array of biomaterials has been conducted, with varying results25,26,27. The aim of cell therapies is to facilitate tissue repair by tissue replacement or the stimulation of local healing through paracrine secretion.<...

Partial colectomy with a primary anastomosis is a commonly performed surgery that can be done for many diseases affecting the colon such as colorectal cancer, Crohn&#39;s disease and diverticulitis1,2. The most dreaded complication after colorectal anastomosis is anastomotic leakage3. Although several risk factors associated with anastomotic leaks have been identified, solutions for preventing leakage remain unkown4,5.
Adipose tissue-derived stromal cells (ASCs) are associated with anti-inflammatory and trophic properties6,7, which makes these cells promising candidates for regenerative therapies8. The effectiveness of ASCs to promote tissue healing was shown in various tissues such as cardiac muscle, skin, and oesophagus9,10,11,12,13. However, there are few reports on the use of ASCs to promote intestinal healing. Local transplantation of ASCs to experimental colorectal anastomoses via ASC-coated biosutures or serosal injections of ASCs showed either no improvement in healing14 or did not prevent anastomotic leakage despite a more favourable anastomotic healing15.
Local transplantation of ASCs in suspension or combined with biomaterials might be associated with insufficient cell retention or an inadequate distribution of transplanted cells11. Cell sheet technology16,17 offers an innovative method of ASC delivery18,19. Therefore, in a previous study, a novel approach was proposed in which ASCs organized in a cell sheet could be applied on experimental colorectal anastomosis20. This study demonstrated that ASC sheet transplantation is successful in reducing colorectal anastomosis leakage after partial colectomy in a rat model. This article reports ASC sheet preparation and surgical transplantation technique.

<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

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

A flow chart of this study depicting both ASC sheet culture and the procedure of colon resection and anastomosis is shown in Figure 1. Figure 2 shows ASC sheet microscopic morphology and the macroscopic appearance of the ASC sheet during and after the detachment. Figure 3 shows the different steps of ASC sheet detachment and transplantation. Figure 4 shows the presence o...

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

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

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

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

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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

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

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

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

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

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