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&#39;s Modified Eagle&#39;s medium (LG-DMEM) with 10 g/L Bovine Serum Albumin (BSA) and 1 g/L collagenase type 1.</li>
	<li>Dissect human subcutaneous abdominal adipose tissue (n = 8, all female, mean age 40 &#177; 9 years old) to small pieces (0.5 cm) using a sterile surgical blade size 10, Adson-Brown tissue forceps and Metzenbaum scissors in a sterile Biological SafetyCabinet.</li>
	<li>Store the dissected tissue in a sterile glass media storage bottle. Weigh the total amount of dissected tissue and start the digestion with the prepared isolation medium (50 g of adipose tissue with 150 mL of isolation medium per bottle). The protocol can be paused here by storing the bottle with the dissected adipose tissue and isolation medium at 4 &#176;C on a roller mixer overnight. Then prewarm the bottle the next day to room temperature 25 &#176;C for 15 min before continuing with the next step. 
	NOTE: Subcutaneous abdominal adipose tissue was obtained as leftover material from donors undergoing breast reconstruction surgery with approval of the Erasmus MC Medical Ethical Committee (# MEC-200) and according to the Code of Conduct: &#34;Proper Secondary Use of Human Tissue&#34; (&#60;http://www.federa.org&#62;). Leftover adipose tissue was only used from donors who did not opt-out to secondary use.</li>
	<li>Digest the dissected adipose tissue in a sterile glass media storage bottle in a shaker-incubator at 150 rpm and 37 &#176;C for 1 h.</li>
	<li>Divide the digested solution into 50 mL tubes and centrifuge the tubes for 10 min at 390 x g.</li>
	<li>Prepare culture medium: LG-DMEM supplemented with 10% fetal bovine serum (FBS), 50 &#181;g/mL gentamicin and 1.5 &#181;g/mL ampothericin B.</li>
	<li>Following the centrifugation, remove the supernatant and resuspend the cell pellet in 20 mL of culture medium (LG-DMEM supplemented with 10% fetal bovine serum (FBS), 50 &#181;g/mL gentamicin and 1.5 &#181;g/mL ampothericin B). Centrifuge the cells again for 5 min at 390 x g and remove the supernatant.</li>
	<li>Resuspend the cell pellet with 10 mL of culture medium (LG-DMEM supplemented with 10% FBS, 50 &#181;g/mL gentamicin and 1.5 &#181;g/mL ampothericin B) and filter the cell suspension through a 100 &#181;m filter.</li>
	<li>Add 50 &#181;L of 3% acetic acid/methylene blue solution (for red blood cell lysis) to 50 &#181;L of cell suspension, mix the solution and use 10 &#181;L to count the cells. Perform cell counting with a hemocytometer. Then plate the cells at a density of 40,000 cells/cm2 in culture medium (LG-DMEM supplemented with 30% FBS, 50 &#181;g/mL gentamicin and 1.5 &#181;g/mL ampothericin B).</li>
	<li>Incubate the cells at 37 &#176;C in a humid atmosphere with 5% CO2 for 24 h.</li>
	<li>Following the incubation, wash the cells with warm (37 &#176;C) phosphate buffered saline (PBS) to remove cell debris and replace the media with 10% FBS culture medium with freshly added ascorbic acid-2-phosphate (25 &#181;g/mL) and human recombinant fibroblast growth factor 2 (FGF2, 1 ng/mL).</li>
	<li>Subculture ASCs at 90% confluence using standard 0.25% trypsin EDTA solution (3 mL for a T175 flask). After 3-5 min, neutralize the 0.25% trypsin EDTA solution with 10 mL of culture medium (LG-DMEM supplemented with 10% FBS, 50 &#181;g/mL gentamicin and 1.5 &#181;g/mL ampothericin B). 
	NOTE: Flow cytometry analysis using common ASC surface markers and tri-lineage differentiation assays were performed previously and revealed that ASCs isolated using this protocol displayed ASC specific characteristics21,22.</li>
	<li>Wash the cells with 10 mL of culture medium (LG-DMEM supplemented with 10% FBS, 50 &#181;g/mL gentamicin and 1.5 &#181;g/mL ampothericin B) for a T175 flask and centrifuge the cells for 8 min at 250 x g. Remove the supernatant and resuspend the cells in 1 mL of culture medium. Count the cells with a hemocytometer.</li>
	<li>Plate the cells in T175 culture flasks at a density of 8,000 cells/cm2.</li>
	<li>Freeze the remaining ASCs in liquid nitrogen with 10% dimethyl sulfoxide (DMSO) in culture medium before further use.</li>
</ol>
2. ASC Sheet Preparation
<ol>
	<li>Pre-coat thermo-responsive culture dishes (3.5 cm diameter) with 1 mL of FBS, then place the dishes in an incubator at 37 &#176;C for at least 30 min before cell seeding.</li>
	<li>Trypsinize ASCs using standard 0.25% trypsin EDTA solution for 3-5 min (3 mL for a T175 culture flask). If there are any cell clumps after trypsinization, filter the cells through a 100 &#181;m filter before counting.</li>
	<li>Neutralize the trypsin solution with LG-DMEM supplemented with 10% FBS (10 mL for a T175 flask) and centrifuge the cell suspension for 8 min at 250 x g. Remove the supernatant and resuspend the cells in 1 mL of LG-DMEM supplemented with 10% FBS.</li>
	<li>After coating the thermo-responsive culture dishes with FBS, move the dishes from the incubator to a warming plate at 37 &#176;C and remove FBS from the dish.</li>
	<li>Seed ASCs at 400,000 cells/cm2 (in total, 3.52 &#215; 106 cells diluted in 2 mL of culture medium per dish).</li>
	<li>Carefully distribute the cells as evenly as possible by gently swinging the dish.</li>
	<li>Culture ASC sheets for 48 h at 37 &#176;C in a humid atmosphere with 5% CO2. 
	NOTE: Try to minimize opening the door of the incubator after seeding ASCs to prevent temperature drops that may interfere with ASC sheet formation or cause premature detachment of formed ASC sheets.</li>
</ol>
3. Partial Colectomy and Colorectal Anastomosis
<ol>
	<li>Induce and maintain Male Wistar rats (weighing between 250-350 g) with 1 L/min of isoflurane (1.5-5%)/oxygen inhalation and inject 0.05 mg/kg buprenorphine intramuscularly.</li>
	<li>Once the animals are under general anesthesia, assess anesthetic depth using reflex testing. Apply vitamin A containing eye ointment to the eyes to prevent dryness. When anesthesia is considered sufficient for surgery, aseptically prepare the abdomen by shaving hairs and spraying the surgical area twice with 70% ethanol. Drape the abdomen with sterile paper drapes. Use an aseptic technique and maintain the sterile field during the whole surgical procedure.</li>
	<li>Make a midline abdominal incision of 5 cm with a sterile surgical blade size 10 and extend the incision with Metzenbaum scissors. Identify and exteriorize the colon and pack the colon off from the remainder of the abdominal cavity with saline moistened gauzes. Identify the right, middle, and left colic arteries in the mesentery, bluntly dissect around each vessel using Halsted mosquito and ligate each individual vessel with non-absorbable braided silk 4/0.</li>
	<li>Isolate the colonic segment between 1.0 cm aborally to the cecum and 0.5 cm above the caudal mesenteric artery. Transect through healthy colon using Metzenbaum scissors. After the resection of the colonic segment, bring the proximal and distal ends of the colon together by introducing two long cotton swabs trans-anally through the distal colon, and then into the proximal colon end.</li>
	<li>Using a surgical microscope, create an insufficiently sutured end-to-end anastomosis by one-layer inverting suturing using 5 interrupted sutures (non-absorbable monofilament polyamide 8/0). Place the interrupted sutures through all layers of the colon wall and position the knots extraluminally.</li>
	<li>Divide the rats randomly into control or ASC sheet group.</li>
</ol>
4. ASC Sheet Transplantation
<ol>
	<li>Allow the culture dishes to cool down to room temperature 40 min before in vivo transplantation, to facilitate ASC sheet detachment. 
	NOTE: After the detachment, ASC sheets shrink to approximately 2 cm in diameter. ASC sheet viability remains high for at least 3-6 h after the detachment20.</li>
	<li>Remove the culture medium and replace it with 1 mL of serum free LG-DMEM.</li>
	<li>Gently grab the rims of the ASC sheet with atraumatic forceps and place the dish-side of the ASC sheet on top of the anastomotic line.</li>
	<li>Carefully stretch out the ASC sheet approximately 0.25 cm above and below the anastomotic line using the atraumatic forceps and wrap the sheet around the colon. Lift the colon up to wrap the ASC sheet around the dorsal side. 
	NOTE: ASC sheets adhere spontaneously to the anastomotic line, there is no need to use tissue glue or any other biomaterial. The control group does not receive any additional treatment.</li>
	<li>Remove the cotton swabs and change the gloves and instruments. Replace the colon in the abdomen. Close the abdomen-linea alba with one layer of running sutures using absorbable braided polyglycolic acid 5/0. Suture the subcutis and the cutis together in one layer of running sutures using absorbable braided polyglycolic acid 5/0.</li>
</ol>
5. Post-operative Evaluation and Follow-up
<ol>
	<li>Immediately rehydrate the animals with a subcutaneous injection of 5 mL warm saline postoperatively. Place the animals under a heat lamp to maintain body temperature and monitor the vital signs until animals regain sufficient consciousness to maintain sternal recumbency. 
	NOTE: Animals that had undergone surgery were not returned to the company of other animals until fully recovered. After recovery, allow free access to water and regular rat chow. For post-surgical pain relief, administer 0.05 mg/kg buprenorphine subcutaneously every 6-8 h for 3 days. No antibiotics were administered at any time in this study.</li>
	<li>Obtain daily clinical evaluations of wellness and weight. In case of a very low wellness score or severe weight loss, animals should be euthanized&#160;by exsanguination via cardiac puncture while still under anesthesia.</li>
	<li>On postoperative day 3 or day 7, anesthetize the rats again with&#160;1 L/min of isoflurane (1.5-5%)/oxygen inhalation without buprenorphine, and perform a re-laparotomy with a U-shaped incision. Check the abdomen for the signs of peritonitis, stricture, fibrous adhesions, and the presence of abscesses. Score the severity of adhesions and abscess both in the abdomen and at the anastomotic area.</li>
	<li>Determine the anastomotic bursting pressure by the insufflation of air in a closed segment of the colon including the anastomosis. Record the air pressure and the place of rupture when the first air leak as bursting pressure.</li>
	<li>Remove the colon from each sacrificed animal and fix the cut segment of the colon -containing the colorectal anastomosis- with 4% buffered formaldehyde, following by paraffin embedding and cutting the colon into 4 &#956;m thick sections.</li>
	<li>Euthanize the animals while under anesthesia directly after collection of the anastomosis segment by exsanguination via cardiac puncture. Confirm animal death with the absence of breathing movement and a heartbeat.</li>
	<li>Perform histochemical stainings such as Hematoxylin &#38; Eosin and Picro Sirius red, and immunohistochemical stainings with antibodies against human mitochondria, rat endothelial cells (anti-CD34) and cells of the immune system (CD3+, CD163+).</li>
	<li>Digitalize the slides for computerized analysis and compare the animal groups.</li>
</ol>

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The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

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.
In this rat model, ASC sheet transplantation was technically successful. Clinical and histological evaluations demonstrated the effectiveness of the ASC sheet at reducing leakage after colon resection with primary anastomosis.
The transplantation of an ASC sheet to colorectal anastomoses is a novel approach in colorectal surgery. In this study, for the first time, a high-density ASC sheet was transplanted. The choice for ASCs delivered as a cell sheet was based on the previously reported advantages of cell sheet technology28,29 over conventional cell transplantation techniques. In addition, the choice for ASCs was based on multiple studies that demonstrated their anti-inflammatory and trophic abilities, especially in cardiac muscle- and skin-healing11,12,19,32.
ASC sheet preparation and detachment can be challenging. In some ASC donors, ASC sheets detached prematurely from the culture dish and folded, precluding their use. In this study, the exact cause for this problem in these specific donors was not identified. Since a high consumption of culture medium was notified in these donors, it was assumed that individual variations in proliferation rate may play an important role. Serum coating assisted in cell adhesion to the surface. Together with careful distribution of the cells in the culture dish and minimizing opening of the incubator door after seeding ASCs (preventing temperature drops), less folding of the ASC sheets occurred. In this way, ASC sheets from all donors were successfully transplanted, confirming the feasibility of the technique.
On the day of the transplantation, most ASC sheets spontaneously detached after allowing the thermo-responsive culture dish to cool down to room temperature. However, a limited number of sheets did not fully detach. Gentle shaking of the culture dish or soft flushing at the rim using a pipette eventually assisted in complete detachments.
For successful ASC sheet transplantation to colorectal anastomoses, several critical steps need to be addressed. First, excessive blood at the anastomosis site should be removed to ensure the contact between the ASC sheet and the serosal surface. Second, the dish side of the ASC sheet should be placed on the serosal surface of the anastomosis. It is postulated that the function of cell sheets after harvest is maintained due to the preservation of cell surface proteins and cell-to-cell junction proteins. Additionally, the ability of ASC sheets to spontaneously adhere to the serosal surface in a short time might be facilitated by the presence of deposited ECM that is produced during in vitro culture19,20,21. Third, the size of the ASC sheet and the colon diameter should be comparable to allow complete coverage of the anastomosis site. When larger surfaces need to be covered, the use of several sheets can be considered.
There are some limitations of this protocol for ASC sheet preparation and transplantation. When using thermo-responsive culture dishes to prepare the ASC sheet, the temperature should strictly be maintained at 37 &#176;C during the entire process to prevent premature detachment. After the transplantation, the ASC sheet survival rate is unknown. Although previous experiments showed that ASCs were viable at 3 days postoperatively, mitochondrial activity of these ASCs was diminished at 7 days postoperatively17. The cell survival rate and dose effect of the transplantation are important questions that need to be addressed in future studies. Observer bias cannot be completely ruled out since the ASC sheet can be identified macroscopically and microscopically at post-operative evaluations. Although transplanted animals did not show more postoperative adhesion formation compared to control animals17, the evaluation of the colorectal anastomosis can be hindered by the presence of these intra-abdominal adhesions.
An advantage of this application technique is that it is simple and easy to perform. With only a small amount of practice and using universal atraumatic forceps, intestinal surgeons should be able to wrap the ASC sheet around the anastomosis. In addition, surgery time is not greatly affected since ASC sheets immediately adhere to host tissues. Moreover, when using ASC sheets, no synthetic biomaterial remains in the body minimizing the risk of a foreign body reaction.
Transplanted ASCs organized in cell sheets demonstrated their ability to reduce colorectal anastomosis leakage. In view of these promising results, future studies should be conducted to evaluate long-term effects both for therapeutic and safety reasons. Besides that, several methods have been described to modify the therapeutic effects of ASC sheets such as inducing overexpression of vascular endothelial growth factor30. These methods may further enhance tissue repair using ASC sheets and should be considered in future studies.

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><tbody><tr><td>LG DMEM</td><td>Gibco, Life technologies</td><td>22320022</td><td>ASC isolation and culture</td></tr><tr><td>Collagenase type I</td><td>Gibco, Life technologies</td><td>17100-01</td><td>ASC Isolation</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td>ASC Isolation</td></tr><tr><td>Fetal bovine serum</td><td>Gibco, Life technologies</td><td>10270-106</td><td>FBS, ASC isolation and culture</td></tr><tr><td>3% acetic acid with methylene blue</td><td>Stemcell Technologies</td><td>7060</td><td>ASC Isolation</td></tr><tr><td>Gentamicin</td><td>Gibco, Life technologies</td><td>15750-037</td><td>ASC isolation and culture</td></tr><tr><td>Ampothericin B</td><td>Gibco, Life technologies</td><td>15290-018</td><td>ASC isolation and culture</td></tr><tr><td>Shaker (Gallenkamp Environmental Shaker Model 10X 400)</td><td>Akribis Scientific</td><td>F240</td><td>ASC Isolation</td></tr><tr><td>Centrifuge</td><td>Hettichlab</td><td>Rotina380</td><td>ASC isolation and culture</td></tr><tr><td>Phosphate buffer saline</td><td>Gibco, Life technologies</td><td>14190-094</td><td>PBS, ASC isolation and culture</td></tr><tr><td>Ascorbic acid-2-phosphate</td><td>Sigma-Aldrich</td><td>A8960</td><td>ASC isolation and culture</td></tr><tr><td>Human recombinant fibroblast growth factor 2</td><td>AbD Serotec</td><td>AF-100-15</td><td>FGF2, ASC isolation and culture</td></tr><tr><td>Trypsin EDTA</td><td>Gibco, Life technologies</td><td>25200-056</td><td>ASC culture</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2650-5x</td><td>DMSO, ASC freezing</td></tr><tr><td>Thermo-responsive culture dishes</td><td>Nunc Thermo scientific</td><td>174904</td><td>ASC sheet preperation</td></tr><tr><td>Non-absorbable braided silk 4/0</td><td>B Braun</td><td>21151042</td><td>surgery</td></tr><tr><td>Non-absorbable monofilament polyamide 8/0</td><td>B Braun</td><td>G1118170</td><td>surgery</td></tr><tr><td>Absorbable braided polyglycolic acid 5/0</td><td>B Braun</td><td>C1048207</td><td>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 of the ASC sheet at the anastomotic line and prevention from leakage 3 days postoperatively.
The follow-up period allowed for colorectal anastomosis evaluation. The different assessments are shown in the figures and table. Compared to control animals, the transplanted animal groups showed less frequent anastomotic dehiscence and leakage on postoperative day 3 and less frequent abscess formation on postoperative day 7. Compared to control animals, transplanted animals did not develop significant adhesions or stricture formation. Table 1 shows the macroscopic findings at the end of the follow up periods in transplanted and non-transplanted animals.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57213/57213fig1.jpg" /> 
Figure 1: Study flow chart. A) ASCs were isolated and expanded before seeding onto thermo-responsive culture dishes. ASCs were seeded at a density of 400,000 cells/cm2 and cultured for 48 h before the transplantation. On the day of transplantation, ASC sheet were detached by allowing the thermo-responsive culture dish to cool down to room temperature for 30 min. B) Scheme of surgical procedure; after ligation of supplying vessels a partial colectomy and colorectal anastomosis is performed. An ASC sheet was wrapped around the anastomosis in the ASC sheet group; a) caecum, b) terminal ileum, c) anus. The schematic overview of the surgical procedure was partly adapted with permission from Wu Z. et al.24 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57213/57213fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57213/57213fig2.jpg" /> 
Figure 2: ASC sheet before and after detachment. A) ASC sheet in thermo-responsive culture dishes after 48 h of culture 40x magnification. ASC sheets were obtained by culturing ASCs on commercial thermo-responsive culture dishes. B-C) ASC sheet during (B) and after detachment (C). When the thermo-responsive culture dishes were allowed to cool down to room temperature, the ASC sheet spontaneously detached from the dish surface as one intact ASC sheet. No enzymatic treatment was necessary. The size of the ASC sheet reduced after the detachment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57213/57213fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57213/57213fig3.jpg" /> 
Figure 3: ASC sheet transplantation. A) Depiction of ASC sheet orientation in the thermo-responsive culture dish.26 B) Orientation of ASC sheet after the transplantation. The dish side of the ASC sheet was placed on top of the serosal surface of the anastomotic line (anastomotic line is indicated with crosses). C) Intra-operative views of the different steps of transplantation. Two atraumatic forceps were used to lift up the ASC sheet and wrap it around the anastomotic line. White arrows indicate ASC sheet location. The images of transplantation were partly adapted with permission from Sukho, P., et al.20 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57213/57213fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57213/57213fig4.jpg" /> 
Figure 4: Colorectal anastomosis macroscopic and histological evaluation. A-B) Compared to the control group, macroscopic observation showed reduced leakage and abscess formation at post-operative day 3 (A) and 7 (B), respectively.White arrows point at anastomotic area and black arrows point at transplanted ASC sheets.C) Representative cross-sections of the colorectal anastomosis site in transplanted animals stained with H&#38;E. The sheet structure could be identified at the anastomosis site up to 7 days postoperatively.The images were partly adapted with permission from Sukho, P., et al.20 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57213/57213fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Post-operative day 3</td>
			<td colspan="3">Post-operative day 7</td>
		</tr>
		<tr>
			<td></td>
			<td>Control (%)</td>
			<td>ASC sheet (%)</td>
			<td>p-value</td>
			<td>Control (%)</td>
			<td>ASC sheet (%)</td>
			<td>p-value</td>
		</tr>
		<tr>
			<td>Peritonitis</td>
			<td>1/14(7.1)</td>
			<td>0/14(0)</td>
			<td>NS</td>
			<td>0/15(0)</td>
			<td>0/14(0)</td>
			<td>NS</td>
		</tr>
		<tr>
			<td>Anastomotic disruption</td>
			<td>10/14(71.4)</td>
			<td>2/14(14.3)</td>
			<td>0.002</td>
			<td>3/15(20)</td>
			<td>2/14(14.3)</td>
			<td>NS</td>
		</tr>
		<tr>
			<td>Stricture</td>
			<td>2/14(14.3)</td>
			<td>2/14(14.3)</td>
			<td>NS</td>
			<td>2/15(13)</td>
			<td>2/14(14.3)</td>
			<td>NS</td>
		</tr>
		<tr>
			<td>Abscess at anastomosis</td>
			<td>14/14(100)</td>
			<td>12/14(85.7)</td>
			<td>NS</td>
			<td>10/15(66.7)</td>
			<td>4/14(28.6)</td>
			<td>0.04</td>
		</tr>
		<tr>
			<td>Adhesion at anastomosis</td>
			<td>14/14(100)</td>
			<td>14/14(100)</td>
			<td>NS</td>
			<td>15/15(100)</td>
			<td>14/14(100)</td>
			<td>NS</td>
		</tr>
		<tr>
			<td>Abscess elsewhere</td>
			<td>11/14(78.5)</td>
			<td>8/14(57.1)</td>
			<td>NS</td>
			<td>6/15(40)</td>
			<td>4/14(28.6)</td>
			<td>NS</td>
		</tr>
		<tr>
			<td>Adhesion elsewhere</td>
			<td>8/14(57.1)</td>
			<td>3/14(21.42)</td>
			<td>NS</td>
			<td>9/15(60)</td>
			<td>7/14(50)</td>
			<td>NS</td>
		</tr>
	</tbody>
</table>
Table 1: Post-operative macroscopic findings.

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 ...

transplantation-adipose-tissue-derived-stem-cell-sheet-to-reduce

Nanyang Technological University

Research

JoVE Journal

Medicine

7.5K Views.  Utrecht University. De voorbereiding en de transplantatie van vetweefsel afgeleid cel van de stam (ASC) bladen op onvoldoende ingehecht colorectal anastomoses in een rat-model wordt gepresenteerd. Deze nieuwe aanvraag blijkt dat ASC bladen colorectal wapendrager lekkage kunnen verminderen.

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

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Cancer Research

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

Bioengineering

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 with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

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

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

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 development of diversified technologies combining adipose tissue-derived stromal cells (ADSCs) with various biomaterials has led to the construction of numerous types of tissue-engineered substitutes, such as bone, cartilage, and adipose tissues from rodent, porcine, or human ADSCs. Extended esophageal endoscopic submucosal dissection (ESD) is responsible for esophageal stricture formation. Stricture prevention remains challenging, with no efficient treatments available. Previous studies reported the effectiveness of mucosal cell-sheet transplantation in a canine model and in humans. ADSCs are attributed anti-inflammatory properties, local immune modulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages. This original study describes the endoscopic transplantation of an ADSC tissue-engineered construct to prevent esophageal stricture in a swine model. The ADSC construct was composed of two allogenic ADSC sheets layered upon each other on a paper support membrane. The ADSCs were labeled with the PKH67 fluorophore to allow probe-based confocal laser endomicroscopy (pCLE) monitoring. On the day of transplantation, a 5-cm and hemi-circumferential ESD known to induce esophageal stricture was performed. Animals were immediately endoscopically transplanted with 4 ADSC constructs. The complete adhesion of the ADSC constructs was obtained after 10 min of gentle application. Animals were sacrificed on day 28. All animals were successfully transplanted. Transplantation was confirmed on day 3 with a positive pCLE evaluation. Compared to transplanted animals, control animals developed severe strictures, with major fibrotic tissue development, more frequent alimentary trouble, and reduced weight gain. In our model, the transplantation of allogenic ADSCs, organized in double cell sheets, after extended ESD was successful and strongly associated with a lower esophageal stricture rate.

This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model.

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

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat.
This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue.
Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat's neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.

This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Biology

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

Experimental Rat Model of Pouchitis for Studying Ulcerative Colitis Pathogenesis and Treatment

A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing lifelong morbidity. When medical treatment is ineffective, especially in cases of massive gastrointestinal bleeding, perforation, toxic megacolon, or carcinogenesis, surgery becomes the last line of defense to cure UC. Total colorectal resection and ileal pouch-anal anastomosis (IPAA) offer the best chance for long-term treatment. Pouchitis is the most common and troublesome postoperative complication. In this investigation, microsurgery is employed to create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat model of pouchitis is established by inducing inflammation of the ileal pouch with dextran sulfate sodium (DSS). The successful establishment of rat pouchitis is validated through analysis of postoperative general status, weight, food and water intake, fecal data, as well as pouch tissue pathology, immunohistochemistry, and inflammatory factor analysis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and treatment of the condition.

Author Spotlight: Developing a Rat Model for Pouchitis Research and Treatment

This protocol describes the method for establishing a rat model of pouchitis. The ileal pouch model was created by performing ileal pouch-anal anastomosis (IPAA) surgery using microsurgical techniques. After the surgery, the rat was treated with 4% dextran sulfate sodium (DSS) for 4 days.

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing ...

Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue

Transfer the harvested adipose tissue into a new 2-milliliter microcentrifuge tube, containing 1 milliliter of freshly prepared digestion medium, and use surgical scissors to mince the tissues. Transfer the tissue slurry into a 50-milliliter tube containing 9 milliliters of fresh digestion medium, and use a 1-milliliter pipette to triturate the tissues 10 times.
When a homogeneous solution has been obtained, incubate the tube for 30 to 45 minutes at 37 degrees Celsius and 100 revolutions per minute, checking every 5 to 10 minutes to prevent over-digestion. When a light yellow homogeneous adipose tissue solution can be observed upon gentle swirling of the tube, stop the digestion with 5 milliliters of HDMEM supplemented with 10% fetal bovine serum. After a thorough mixing, centrifuge the adipose tissue sample.
The stromal vascular cell fraction will be visible as a brownish pellet. Resuspend the pellet in 10 milliliters of culture medium, and filter the cell suspension through a 70-micrometer cell strainer.
Collect the cells by another centrifugation, and gently resuspend the pellet in 5 milliliters of erythrocyte lysis buffer. Transfer the suspension to a 15-millimeter tube. After 10 minutes, stop the reaction with two centrifugations in 10 milliliters of PBS, supplemented with 1% fetal bovine serum.
After the second centrifugation, resuspend the pellet in 5 milliliters of culture medium on ice, and collect the cells with a final centrifugation. Then, resuspend the cells in 5 milliliters of FACS buffer for counting.
To set up an NCADSC culture after their isolation by FACS according to standard protocols, place the cells at a 5 x 103 cells per cm2 concentration in each well, a 12-well culture plate in complete culture medium, at 37 degrees Celsius, and 5% carbon dioxide for 20 to 24 hours. The next day, wash the cells with 37 Celsius PBS, and feed the culture with fresh culture medium.

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

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