Name: creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model
Uploaded: 2017-08-04T12:30:00
Description: Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

The authors thank Dr. Yukiko Koga of the Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, for providing practical advice. We also thank Mr. Hidekazu Murata of the Diabetic Center of Tokyo Women&#8217;s Medical University School of Medicine for excellent technical support. This study was supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program of the Project for Developing Innovation Systems &#8220;Cell Sheet Tissue Engineering Center (CSTEC)&#8221; from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

All experimental protocols presented below were approved by the Animal Welfare Committee of Tokyo Women&#39;s Medical University School of Medicine and abided by all requirements of the Guidelines for Proper Conduct of Animal Experiments.
1. Preparation of Animals, Instruments, Culture Media, and Dishes
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	<li>Prepare complete culture medium using minimum essential medium alpha containing 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Store this for several months at 4 &#176...

<ol>
	<li>Boulton, A. J., Vileikyte, L., Ragnarson-Tennvall, G., Apelqvist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+diabetic+foot+disease.">The global burden of diabetic foot disease.</a> Lancet. 366 (9498), 1719-1724 (2005).</li><li>Zannettino, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotential+human+adipose-derived+stromal+stem+cells+exhibit+a+perivascular+phenotype+in+vitro+and+in+vivo.">Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo.</a> J Cell Physiol. 214 (2), 413-421 (2008).</li><li>Kern, S., Eichler, H., Stoeve, J., Kluter, H., Bieback, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+mesenchymal+stem+cells+from+bone+marrow,+umbilical+cord+blood,+or+adipose+tissue.">Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.</a> Stem Cells. 24 (5), 1294-1301 (2006).</li><li>Casteilla, L., Planat-Benard, V., Laharrague, P., Cousin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stromal+cells:+Their+identity+and+uses+in+clinical+trials,+an+update.">Adipose-derived stromal cells: Their identity and uses in clinical trials, an update.</a> World J Stem Cells. 3 (4), 25-33 (2011).</li><li>Zuk, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Eng. 7 (2), 211-228 (2001).</li><li>Zuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The ASC: Critical Participants in Paracrine-Mediated Tissue Health and Function. , (2013).</li><li>Nie, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+administered+adipose-derived+stem+cells+accelerate+wound+healing+through+differentiation+and+vasculogenesis.">Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis.</a> Cell Transplant. 20 (2), 205-216 (2011).</li><li>Shin, L., Peterson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cell+grafts+enhance+normal+and+impaired+wound+healing+by+recruiting+existing+endogenous+tissue+stem/progenitor+cells.">Human mesenchymal stem cell grafts enhance normal and impaired wound healing by recruiting existing endogenous tissue stem/progenitor cells.</a> Stem Cells Transl Med. 2 (1), 33-42 (2013).</li><li>Okano, T., Yamada, N., Sakai, H., Sakurai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+recovery+system+for+cultured+cells+using+plasma-treated+polystyrene+dishes+grafted+with+poly(N-isopropylacrylamide).">A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide).</a> J Biomed Mater Res. 27 (10), 1243-1251 (1993).</li><li>Yamato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-responsive+culture+dishes+allow+the+intact+harvest+of+multilayered+keratinocyte+sheets+without+dispase+by+reducing+temperature.">Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature.</a> Tissue Eng. 7 (4), 473-480 (2001).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+cell+sheet+transplantation+improves+damaged+heart+function+via+superior+cell+survival+in+comparison+with+dissociated+cell+injection.">Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection.</a> Tissue Engineering Part A. 17 (23-24), 2973-2980 (2011).</li><li>Kuhlmann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramyocellular+lipid+and+insulin+resistance:+a+longitudinal+in+vivo+1H-spectroscopic+study+in+Zucker+diabetic+fatty+rats.">Intramyocellular lipid and insulin resistance: a longitudinal in vivo 1H-spectroscopic study in Zucker diabetic fatty rats.</a> Diabetes. 52 (1), 138-144 (2003).</li><li>Slavkovsky, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zucker+diabetic+fatty+rat:+a+new+model+of+impaired+cutaneous+wound+repair+with+type+II+diabetes+mellitus+and+obesity.">Zucker diabetic fatty rat: a new model of impaired cutaneous wound repair with type II diabetes mellitus and obesity.</a> Wound Repair Regen. 19 (4), 515-525 (2011).</li><li>Oltman, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+vascular+and+neural+dysfunction+in+sciatic+nerves+of+Zucker+diabetic+fatty+and+Zucker+rats.">Progression of vascular and neural dysfunction in sciatic nerves of Zucker diabetic fatty and Zucker rats.</a> Am J Physiol Endocrinol Metab. 289 (1), E113-E122 (2005).</li><li>Coppey, L. J., Gellett, J. S., Davidson, E. P., Dunlap, J. A., Yorek, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+endoneurial+blood+flow,+motor+nerve+conduction+velocity+and+vascular+relaxation+of+epineurial+arterioles+of+the+sciatic+nerve+in+ZDF-obese+diabetic+rats.">Changes in endoneurial blood flow, motor nerve conduction velocity and vascular relaxation of epineurial arterioles of the sciatic nerve in ZDF-obese diabetic rats.</a> Diabetes Metab Res Rev. 18 (1), 49-56 (2002).</li><li>Galiano, R. D., Michaels, V., Dobryansky, M., Levine, J. P., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+reproducible+murine+model+of+excisional+wound+healing.">Quantitative and reproducible murine model of excisional wound healing.</a> Wound Repair Regen. 12 (4), 485-492 (2004).</li><li>Lin, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+multi-layer+adipose-derived+stem+cell+sheet+in+a+full-thickness+wound+healing+model.">Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model.</a> Acta Biomater. 9 (2), 5243-5250 (2013).</li><li>McLaughlin, M. M., Marra, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+adipose-derived+stem+cells+as+sheets+for+wound+healing.">The use of adipose-derived stem cells as sheets for wound healing.</a> Organogenesis. 9 (2), 79-81 (2013).</li><li>Cerqueira, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+stem+cells+cell+sheet+constructs+impact+epidermal+morphogenesis+in+full-thickness+excisional+wounds.">Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds.</a> Biomacromolecules. 14 (11), 3997-4008 (2013).</li><li>Koga, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+course+of+full-thickness+skin+defects+with+exposed+bone:+an+evaluation+by+a+quantitative+examination+of+new+blood+vessels.">Recovery course of full-thickness skin defects with exposed bone: an evaluation by a quantitative examination of new blood vessels.</a> J Surg Res. 137 (1), 30-37 (2007).</li><li>Cianfarani, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+impairs+adipose+tissue-derived+stem+cell+function+and+efficiency+in+promoting+wound+healing.">Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing.</a> Wound Repair Regen. 21 (4), 545-553 (2013).</li><li>Matsuda, K., Suzuki, S., Isshiki, N., Ikada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-freeze+dried+bilayer+artificial+skin.">Re-freeze dried bilayer artificial skin.</a> Biomaterials. 14 (13), 1030-1035 (1993).</li><li>Miyahara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayered+mesenchymal+stem+cells+repair+scarred+myocardium+after+myocardial+infarction.">Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction.</a> Nat Med. 12 (4), 459-465 (2006).</li><li>Iwata, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+engineering+and+its+application+for+periodontal+regeneration.">Cell sheet engineering and its application for periodontal regeneration.</a> J Tissue Eng Regen Med. , (2013).</li><li>Elloumi-Hannachi, I., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+engineering:+a+unique+nanotechnology+for+scaffold-free+tissue+reconstruction+with+clinical+applications+in+regenerative+medicine.">Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine.</a> J Intern Med. 267 (1), 54-70 (2010).</li><li>Watanabe, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+adipose+tissue-derived+stem/stromal+cells,+using+simian+immunodeficiency+virus-based+lentiviral+vectors,+in+the+treatment+of+hemophilia.">Genetically modified adipose tissue-derived stem/stromal cells, using simian immunodeficiency virus-based lentiviral vectors, in the treatment of hemophilia.</a> B. Hum Gene Ther. 24 (3), 283-294 (2013).</li><li>Kim, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+effect+of+adipose-derived+stem+cells:+a+critical+role+of+secretory+factors+on+human+dermal+fibroblasts.">Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts.</a> J Dermatol Sci. 48 (1), 15-24 (2007).</li><li>Nakagami, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+autologous+cell+therapy+in+ischemic+limb+disease+through+growth+factor+secretion+by+cultured+adipose+tissue-derived+stromal+cells.">Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells.</a> Arterioscler Thromb Vasc Biol. 25 (12), 2542-2547 (2005).</li><li>Asahara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+contributes+to+postnatal+neovascularization+by+mobilizing+bone+marrow-derived+endothelial+progenitor+cells.">VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.</a> EMBO J. 18 (14), 3964-3972 (1999).</li><li>Kato, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+transplantation+of+an+adipose-derived+stem+cell+(ASC)+sheet+combined+with+artificial+skin+accelerates+wound+healing+in+a+rat+wound+model+of+type+2+diabetes+and+obesity.">Allogeneic transplantation of an adipose-derived stem cell (ASC) sheet combined with artificial skin accelerates wound healing in a rat wound model of type 2 diabetes and obesity.</a> Diabetes. , db141133 (2015).</li></ol>

The most critical steps for successfully culturing an rASC sheet are as follows: 1) The temperature must be maintained at approximately 37 &#176;C during culturing on the temperature-responsive culture dishes. During the creation of an rASC sheet, every procedure was performed on a 37 &#176;C thermo-plate, and every reagent was warmed to 37 &#176;C to prevent the cells from spontaneously detaching from the dish31. 2) The recipient ZDF rats must be monitored to prevent the removal of the non-adhesi...

The population of diabetic patients is increasing worldwide and reached 400 million in 20151; an estimated 15 - 25% of patients with diabetes are at risk from the progression of a lower-extremity diabetic ulcer2. Lower-extremity diabetic ulcers are intractable and might require a prolonged therapeutic period with rehabilitation training after complete recovery. A long therapy period often results in a significant reduction in patient quality of life. Thus, new therapies that decrease or prevent aggravation must be developed for the treatment of diabetic wounds. To evaluate diabetic wound healing, we optimized a diabetic ulcer wound-healing model in rats, which mimics practical clinical conditions, and evaluated whether transplanting adipose-derived stem cell (ASC) sheets using cell-sheet engineering accelerated wound healing.
Mesenchymal stromal cells (MSCs) exhibit an excellent potential for accelerating wound healing because of their self-renewal capacity, their immunomodulatory effects, and their ability to differentiate into various cell lineages3. ASCs are a type of MSC derived from adipose tissue, and they exhibit several advantages over MSCs derived from other tissues, including their angiogenic potential and paracrine activity4,5. Adipose tissue is relatively abundant in the human body, and its accessibility allows for collection using minimally invasive procedures. Therefore, ASCs have been used experimentally for wound-healing applications6,7.
Previous reports have shown that the direct injection of single-cell MSC suspensions into areas around wounds can accelerate wound healing8,9. However, despite reports of the acceleration of wound healing in diabetic ulcer models following the injection of single-cell suspensions, the survival time of transplanted cells at the wound site is not clear.
In this study, we applied cell-sheet engineering using temperature-responsive culture dishes. These dishes have the temperature-responsive polymer N-isopropylacrylamide covalently bound onto their surface10. The grafted polymer layer allows for temperature-controlled cell adhesion to or detachment from the surface of the culture dish. The surface of the dish becomes hydrophobic at 37 &#176;C, allowing cells to adhere and proliferate, whereas cells spontaneously detach from the surface when it becomes hydrophilic at temperatures below 32 &#176;C. Cultured cells can be harvested as a contiguous cell sheet with intact cell-to-cell junctions and extracellular matrices (ECMs) simply by reducing the temperature; thus, proteolytic enzymes that damage the ECM, such as trypsin, are not required11. Therefore, cell-sheet engineering can preserve cell-to-cell connections and improve the efficacy of cell transplantation.
In addition, cell-sheet transplantation increases cell survival rates when compared to cell injection12.In this protocol, Zucker diabetic fatty (ZDF) rats were selected as a type 2 diabetes and obesity model with delayed wound healing. ZDF rats spontaneously develop obesity at approximately 4 weeks. They then develop type 2 diabetes with obesity between 8 and 12 weeks of age, at which point they exhibit hyperglycemia associated with insulin resistance, dyslipidemia, and hypertriglyceridemia13. Delayed wound healing, reduced blood flow in peripheral blood vessels, and diabetic nephropathy are also observed14,15,16. Moreover, ZDF rats might be an appropriate model for studying the healing of intractable cutaneous ulcers, such as diabetic ulcers.
The differences between humans and rodents in wound-healing mechanisms are associated with anatomical differences in the skin. Wound healing in normal rats is based on wound contraction, whereas wound healing in humans is based on re-epithelialization and granulation tissue formation. Typically, wound splinting used in rodent models helps to minimize wound contraction and allows for the gradual formation of granulation tissue17, although wounds in nondiabetic rats are almost completely closed by contraction. However, diabetic wound contraction in ZDF rats is impaired, and wound healing primarily occurs through re-epithelialization and granulation tissue formation; thus, this process is more similar to human wound healing14.
Diabetic wounds with exposed bone after debridement are often encountered clinically. Previous studies have examined 12-mm diameter full-thickness skin wounds on the backs of athymic nude mice18,19 and 10-mm diameter full-thickness skin wounds on the backs of normal mice20. To develop a clinical model for severe diabetic wounds, larger (15 x 10 mm2) full-thickness skin defects with exposed bone and without the periosteum were created, as previously described21, in rats with type 2 diabetes and obesity.
Rat ASC (rASC) sheets from the ASCs of normal Lewis rats were created through the allogenic transplantation of ASC sheets. In clinical practice, autologous transplantation is unfeasible because diabetic patients with ulcers often exhibit severe diabetic complications, such as uncontrolled high blood glucose levels and high body mass indices, and these complications cause wound-healing disorders that increase the difficulty of obtaining adipose tissue from these patients. Furthermore, ASCs from animals with diabetes exhibit altered properties and impaired function22. Therefore, the protocol presented here describes the allogenic transplantation of rASC sheets from normal rats and the application of artificial skin to diabetic rats.
The bilayer artificial skin used in this protocol prevents the spontaneous contraction of the wounds, promotes the synthesis of a new connective tissue matrix, and resembles the true dermis23. In this protocol, artificial skin is placed on an rASC sheet and fixed with nylon threads to prevent wound contraction or enlargement resulting from loose rat skin. In addition, the artificial skin provides a three-dimensional framework for the ASC sheets, maintains a moist environment for the transplanted ASC sheets and wounds, and protects the wounds from infection and external forces. Finally, a non-adhesive dressing is placed over the wound to protect it from external impact, maintain a moist wound environment, and absorb exudate.
An rASC sheet is thin, flexible, and deformable and can be adhered to moving recipient sites, such as a beating heart24. Cell-sheet engineering has been used for the reconstruction of various tissues and can generate curative effects25,26. ASC sheets that exhibit clinical therapeutic potential might accelerate the healing of many types of wounds. Moreover, the allogeneic transplantation of ASC sheets, combined with the use of artificial skin, might be applicable to the treatment of intractable ulcers or burns, such as those observed in peripheral arterial disease or collagen disease, or they may be administered to patients who are undernourished or are using steroids. This approach increases the efficiency of transplanting ASCs. The wound-healing ZDF rat model produces a severe wound condition that resembles the human wound healing process and mimics clinical conditions in a small-sized experimental animal.

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			<td height="21" style="height:21px;width:223px;">&#945;-MEM glutamax</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:119px;">32571-036</td>
			<td style="width:167px;">Carlsbad, CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Japan Bioserum Co Ltd.</td>
			<td style="width:119px;">S1650-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">10 103 578 001</td>
			<td>Mannheim, Germany</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">60-cm2 Primaria tissue culture dish</td>
			<td>BD Biosciences</td>
			<td>353803</td>
			<td>Franklin Lakes, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate Buffer Saline (PBS)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">1490-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-ascorbic acid phosphate magnesium salt n-hydrate</td>
			<td style="width:169px;">Wako</td>
			<td>013-19641</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">35-mm temperature-responsive culture dish (UpcellTM)</td>
			<td>CellSeed</td>
			<td>NUNC-174904</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microwarm plate (MP-1000)</td>
			<td style="width:169px;">Kitazato Science Co., Ltd.</td>
			<td style="width:119px;">1111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rodent mechanical ventilator</td>
			<td>Stoelting</td>
			<td>#50206</td>
			<td>Wood Dale, IL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% isoflurane</td>
			<td>Pfizer Japan</td>
			<td>114-13340-3</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Artificial skin (Pelnac&#174;)</td>
			<td>Smith &#38; Nephew</td>
			<td>PN-R40060</td>
			<td>&#160;Tokyo, Japan</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Non-adhesive dressing (Hydrosite plus&#174;)</td>
			<td>Smith &#38; Nephew</td>
			<td align="right">66800679</td>
			<td>Known as Allevyn non-adhessing&#174; in the United State</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">5-0 nylon suture</td>
			<td>Alfresa</td>
			<td style="width:119px;">EP1105NB45-KF2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 CELLSTAR TUBES</td>
			<td>greiner bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15mL Centrifuge Tube</td>
			<td>Corning Incorporated</td>
			<td align="right" style="width:119px;">430791</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">14 GOLDMAN-FOX PERIOSTEAL</td>
			<td>Hu-Friedy</td>
			<td style="width:119px;">P14</td>
			<td>Chicago, IL</td>
		</tr>
	</tbody>
</table>

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.

This protocol attempted to establish a new cell-based therapy for intractable diabetic wounds. Briefly (as illustrated in Figure 1), allogeneic rASC sheets were created from normal rats using cell-sheet engineering and were then transplanted using a bilayer of artificial skin onto a full-thickness skin defect on a diabetic rat. Light microscope images of a good example of an rASC sheet (Figure 2A) and a bad example of an rASC sheet (Figure 2B</str...

Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

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.

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 ...

Research

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Medicine

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