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.<...

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.

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			<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<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>
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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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7.5K Views.  Utrecht University. 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.

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

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

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

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 protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

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

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

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

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

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

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

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

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 ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

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 ...

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 conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

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

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

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

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

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