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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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 ...

Because they assume positive effect on tissue healing ASC sheets have been tested in many disease model, such as, myocardial infarction. This experiment show that ASC sheets can be used intra-abdominally and apply to intestine. The main advantage of ASC sheets is that no synthetic materials are needed.In addition, ASC sheet transplantation is relatively easy to perform, with only a small amount of practice. This novel application of ASC sheet transplantation was developed to prevent leakage after bowel resection, and offers a first step to which a new therapeutic strategy. ASC sheets applied to intestinal anastomosis have been shown to be promising in preventing leakage.ASC sheets may also be applied to other intra-abdominal organs, such as, the urinary tract. Begin by dissecting human subcutaneous abdominal adipose tissue into 0.5 cm pieces, with a size 10 sterile surgical blade, Adson-Brown tissue forceps and Metzenbaum scissors. Weigh the total amount of dissected tissue in a sterile glass bottle.Add 30 ml of isolation medium per bottle. And digest the dissected adipose tissue in a shaker incubator at 150 rpm and 37 degrees Celsius for one hour. After an hour divide the digested tissue solution into 50 ml tubes, and centrifuge the tubes for 10 minutes at 390 G.Following the centrifugation remove the supernatant.Add medium to resuspend the cell pellets, and add more culture medium, up to 45 ml. Centrifuge the combined cells for five minutes at 390 G and remove the supernatant. Resuspend the cell pellet with 10 ml of culture medium.And filter the cell suspension through a 100 micron filter. Add 50 l of 3%acidic acid methylene blue solution, to 50 l of cell suspension, to lice red blood cells. After mixing the solution, pipette 10 l onto a hemocytometer, and count the cells.Plate the cells at a density of 40000 cells per square cm, in a T175 flask containing culture medium. And incubate the cells at 37 degrees Celsius in a humid atmosphere with 5%CO2 for 24 hours. The next day, wash the cells with warm phosphate buffered saline to remove cell debris.Then, replace the medium with 10%FBS culture medium, plus freshly added ascorbic acid-2-phosphate and human recombinant fibroblast growth factor two. Incubate until the cells reach 90%confluence. Subculture the adipose stem cells at 90%confluence, using standard 0.25%Trypsin-EDTA solution.After three to five minutes neutralize the 0.25%Trypsin-EDTA solution with 10 ml of culture medium. Transfer to a centrifuge tube, and centrifuge the cells for eight minutes at 250 G.Remove the supernatant, and resuspend the cells in 1 ml of culture medium. After counting the cells with a hemocytometer, plate the cells in T175 culture flasks, at a density of 8000 cells per square cm.Freeze the remaining ASCs in liquid nitrogen, with 10%dimethyl sulfoxide in culture medium before further use. Precoat 3.5 cm diameter thermo-responsive culture dishes, with 1 ml of fetal bovine serum. Then place the dishes in an incubator at 37 degrees Celsius, for at least 30 minutes before seeding.To assist ASC adhesion to the thermo-responsive dish, and reduce the risk of premature ASC sheet detachment in culture, the thermo-responsive dish should be precoat with fetal bovine serum before cell seeding. Trypsinize ASCs as before, if there are any cell clumps after trypsinization, filter the cells through a 100 micron filter before counting. Remove the dishes from the incubator, and transfer to a warming plate at 37 degrees Celsius.Remove FBS from the dish. After resuspending the cell pellet in LG DMEM, with 10%FBS, and counting the cells, dilute 3.52 times 10 to the 6th ASCs in 2 ml of culture medium per dish, to seed 400000 cells per square cm in each 3.5 cm dish. Carefully distribute the cells as evenly as possible, by gently swinging the dish.Then, culture ASC sheets for 48 hours at 37 degrees Celsius in a humid atmosphere with 5%CO2. On the day of transplantation, allow the culture dishes to cool to room temperature 40 minutes before in vivo transplantation to facilitate ASC sheet detachment. Once cooled, remove the culture medium, and replace it with 1 ml of serum-free LG DMEM.To transplant the cell sheet gently grab the edge of ASC sheet with atraumatic forceps and place the dish side of the ASC sheet on top of the anastomotic line. Gentle sheet handling and correct sheet placement are essential to ensure successful intact sheet transplantation. Carefully stretch out the ASC sheet to approximately 0.25 cm above and below the anastomotic line.Wrap the sheet around the colon, lifting the colon, to wrap the ASC sheet around the dorsal side. Control animals, that did not receive transplanted ASC sheets, exhibit fecal leakage near the anastomotic line, indicated by the white arrow at post-operative day three. Those animals with transplanted ASC sheets, indicated by the black arrow, showed reduced fecal leakage and abscess formation at post-operative day three.The same result was observed seven days after surgery. This image shows a representative cross-section of the colorectal anastomosis site in a transplanted animal stained with H and E.The sheet structure could be identified at the anastomosis site, up to seven days post-operatively. Once mastered, this technique can be done in a few minutes if it is performed properly.ASC sheet will be spontaneously adherent to the zero cell surface of the colon, there is no need for using glue or suture to fit the ASC sheet. After watching this video you should have a good understanding of how to prepare and transplant ASC sheets onto colorectal anastomosis in rats, for preventing leakage after partial colectomy.

Research

JoVE Journal

Medicine

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