作者感谢塑料部和整形外科手术，东京大学医学院博士由纪子古贺提供实用的建议。我们还感谢先生秀和村田的糖尿病中心的东京妇女医疗大学医学院为优异的技术支持。这项研究被支持由创新中心创造先进的跨学科研究领域项目的发展中国家创新系统"单元格表组织工程中心 (CSTEC)"项目从部的教育、 文化、 体育、 科学和技术 （文部科学省） 的日本。

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
<ol>
	<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;C until use.</li>
	<li>Use adult male ZDF rats as a type 2 diabetic obesity model. Use the fat pad of male Lewis rats to isolate adipose tissue to prepare the cell sheets.</li>
</ol>
2. Isolation and Culture of rASCs
<ol>
	<li>Obtain adipose tissue from Lewis rats (8 - 33 weeks old, male) under general anesthesia using isoflurane.
	<ol>
		<li>Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauzes, a scalpel, a needle holder, operating scissors, a pair of forceps, and a 5-0 nylon suture. Sterilize the surgical instruments and supplies.</li>
		<li>Prepare a 100-mm Petri dish with 10 mL of sterile phosphate-buffered saline (PBS) to temporarily preserve the obtained adipose tissue. Weigh the Petri dish with PBS using a balance before starting surgery to measure the collected adipose tissue. Lay out the surgical instruments and supplies on a sterile drape.</li>
		<li>Anesthetize the rats by using isoflurane at 3 - 4% via a rodent mechanical ventilator and maintain the rats at 2 - 3% isoflurane during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat.</li>
		<li>While wearing sterile gloves, position the rat in a supine position on a sterile drape. Shave the operative area with an electric razor and clean the abdominal section of the rat using sterile gauze soaked in 70% ethanol.</li>
		<li>Create an approximately 5 cm-long skin incision with a scalpel in the lateral lower abdomen of the rat. Expose and dissect the external oblique muscle. Then, expose the epididymal adipose tissue surrounding the epididymis. Gently pull the epididymal adipose tissue out. 
		NOTE: The adipose tissue can be obtained on either side of the rat&#39;s abdomen.</li>
		<li>Excise only the epididymal adipose tissue, avoiding damage to the epididymis, testis, and epididymal blood vessels (Figure S1). Soak the excised adipose tissue in the PBS in the Petri dish to prevent dryness and weigh the tissue.</li>
		<li>Close the abdominal muscle with 5-0 VICRYL and the skin with a 5-0 nylon suture. Then, return each rat that has undergone surgery to a separate cage (one rat per cage). 
		NOTE: Monitor the rats until they fully recover.</li>
	</ol>
	</li>
	<li>Isolate rASCs from 2 - 3 g of adipose tissue (excised from a Lewis rat and processed according to a previously-reported method)27. Briefly, enzymatically digest the excised adipose tissue with 0.1% type A collagenase at 37 &#176;C for 1 h to obtain the stromal-vascular fraction (SVF; Figure S2).
	<ol>
		<li>Prepare several 100-&#181;m cell strainers, several 50-mL tubes, several 15 mL tubes, and two scalpels on a biological clean bench. Turn on a centrifuge and warm up a water bath to 37 &#176;C. In addition, prepare approximately 50 mL of sterile PBS in a 50 mL tube with 0.1% (final concentration) of type A collagenase.</li>
		<li>Mince the adipose tissue into small pieces using two scalpels.</li>
		<li>Move all minced adipose tissue to a 50-mL tube and add PBS to bring the total volume to 15 mL.</li>
		<li>Rest the 50-mL tube upright for 5 min. Discard the upper layer and collect the lower layer except for the debris.</li>
		<li>Add 0.1% of type A collagenase solution to the 50-mL tube with PBS containing the upper layer in order to enzymatically digest the adipose tissue. 
		NOTE: Warm approximately 20 mL of PBS per 2 - 3 g of adipose tissue in a 37 &#176;C water bath.</li>
		<li>Tilt and shake the 50 mL tube gently at 120 - 130 rpm for 1 h in a 37 &#176;C water bath.</li>
		<li>Rest the 50 mL tube upright for 5 min after shaking.</li>
		<li>Filter the solution using a 100 &#181;m cell strainer and pour the solution into a new 50 mL tube. Dispense the filtered solution into two 15 mL tubes.</li>
		<li>Centrifuge the solution in the 15 mL tubes for 5 min at 700 x g. Carefully remove the upper layer of supernatant and leave 5 mL of the lower layer of the solution. Do not aspirate the pellet.</li>
		<li>Add approximately 5 mL of complete culture medium to each 15 mL tube, up to 10 mL per 15 mL tube, and carefully re-suspend the pellet.</li>
		<li>Repeat steps 2.2.8 - 2.2.9. Then, obtain the SVF.</li>
	</ol>
	</li>
	<li>Prepare two 60 cm2 culture dishes. Collect the SVF after two centrifugations at 700 &#215; g for 5 min. Suspend the SVF and plate 10 mL of SVF solution on each 60-cm2 culture dish.</li>
	<li>Culture the dishes for 24 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Prepare PBS and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA), storable for several months at 4 &#176;C until use.</li>
	<li>After initial plating (24 h), wash the cells with PBS 3 times to remove unattached cells and debris. Add fresh complete medium.</li>
	<li>Passage the cells that are nearly sub-confluent on day 3. Add 1 mL of 0.25% trypsin-EDTA and incubate the cells at 37 &#176;C in a 5% CO2 incubator for 3 - 5 min. 
	NOTE: Complete the trypsinization step within 10 min to prevent cell damage.</li>
	<li>Observe the cells under a light microscope after trypsinization to confirm that all the cells have thoroughly detached from the dishes. Then, add 9 mL of complete culture medium to the dishes.</li>
	<li>Count the number of cells using a hemocytometer.</li>
	<li>Subculture the cells at a density of 1.7 x 103 cells/cm2 every 3 days until passage 3. Culture the subcultured cells at 37 &#176;C in the humidified atmosphere of a 5% CO2 incubator.</li>
</ol>
3. Creation of rASC Sheets
<ol>
	<li>Prepare several 35-mm diameter temperature-responsive culture dishes. Pre-coat the dishes with FBS (or complete medium containing FBS) at 37 &#176;C in a 5% CO2 incubator for more than 1 h. 
	NOTE: A 35 mm diameter temperature-responsive culture dish is a commercially available product.</li>
	<li>Prepare a thermo-plate and incubate at 37 &#176;C for the following experimental procedures. 
	NOTE: Perform every procedure using temperature-responsive culture dishes on a 37 &#176;C thermo-plate to prevent the cells from spontaneously detaching from the dish.
	<ol>
		<li>Warm the complete culture medium used in the procedures to 37 &#176;C prior to experimentation.</li>
		<li>Seed passage 3 rASCs derived from Lewis rats onto a 35 mm diameter temperature-responsive culture dish at a density of 1.5 x 105 cells/dish for 3 days using a 2 mL total volume of complete culture medium. 
		NOTE: When rASCs are plated onto a new culture dish, the dish should be slowly rocked back and forth and left and right in an incubator to achieve uniform rASC seeding and a uniformly thick rASC sheet.</li>
		<li>Change 2 mL of the medium to 2 mL of complete culture medium containing 16.4 &#181;g/mL L-ascorbic acid phosphate magnesium salt n-hydrate (AA) on day 3 after seeding to the temperature-responsive culture dishes incubated on a 37 &#176;C thermo-plate. 
		NOTE: Dissolved AA can be stored at -30 &#176;C for months.</li>
		<li>Culture the cells for an additional 4 - 5 days and replace the medium every 2 days with fresh medium containing AA.</li>
		<li>Confirm the proliferation and generation of ASCs under a light microscope to determine whether gaps occurred between the cells in a contiguous cell sheet.</li>
	</ol>
	</li>
	<li>Transfer the cell sheets from the incubator to the benchtop for 15 - 20 min to cool them to room temperature.; observe the cells spontaneously detach as a contiguous cell sheet. Harvest the rASC sheet from the dish surface with a pair of forceps. 
	NOTE: Usually, rASC sheets can be handled with a pair of forceps. If necessary, a transfer membrane can be used to transfer a cell sheet from the culture dish to the wound site if the cell sheet is brittle and fragile.</li>
</ol>
4. Preparation of the Full-thickness Skin Defect Wound Model and Transplantation of rASC Sheets
<ol>
	<li>Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauze, 5-0 nylon suture, a scalpel, a periosteal raspatory, a needle holder, operating scissors, and a pair of forceps. Sterilize the surgical instruments and supplies. Lay out the surgical instruments and supplies on a sterile drape.</li>
	<li>Prepare PBS and maintain it at room temperature, along with some artificial skin and a non-adhesive dressing. Precut the artificial skin (15 x 10 mm) and non-adhesive dressing (20 &#215; 15 mm). Soak the inner collagen sponge layer of the artificial skin in saline before using the artificial skin. 
	NOTE: The artificial skin is made of two layers: an outer silicone sheet layer and an inner collagen sponge.</li>
	<li>Use ZDF rats (16 - 18 weeks old, male, 500 - 600 g) as a wound-healing model for type 2 diabetes and obesity.</li>
	<li>Anesthetize the ZDF rats by inhalation of 4% isoflurane using a rodent mechanical ventilator. Induce anesthesia by using isoflurane at 4 - 5% via a rodent mechanical ventilator and maintain it at 3 - 4% during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat via a toe pinch.</li>
	<li>While wearing sterile gloves, position the rat in the prone position on a sterile drape. Clean the head of the rat using sterile gauze soaked in 70% ethanol, shave the operative area with an electric razor, and clean the skin after shaving using sterile gauze soaked in 70% ethanol.</li>
	<li>Create a squared full-thickness skin defect (15 x 10 mm2) on the head of the anesthetized ZDF rats by removing the cutaneous tissue from the epidermis to the periosteum. Excise the skin and cutaneous tissue with a scalpel and remove the periosteum with a periosteal raspatory.</li>
	<li>Move the rASCs on the 35 mm temperature-responsive culture dish from the incubator to a room-temperature environment immediately prior to transplantation.</li>
	<li>Observe the rASCs on the 35 mm temperature-responsive culture dish spontaneously detach as a sheet from the dish surface after the formation of a wound. Harvest the rASC sheets after 7 days of culturing on temperature-responsive culture dishes. Remove the cultured medium and wash the rASC sheet with PBS three times. Soak the rASC sheet in PBS until transplantation to prevent desiccation.</li>
	<li>Place the rASC sheet immediately over the skull defect using a pair of forceps. If the cell sheet is brittle and fragile, a membrane can be used as a scaffold for transferring the cell sheet from the culture dish to the wound site.</li>
	<li>Cover the rASC sheet and defect with the artificial skin (15 x 10 mm2) and close the wound with approximately 10 stiches using the 5-0 nylon suture. To protect the wound, place a non-adhesive dressing (20 x 15 mm2) over the artificial skin, using 5-0 nylon sutures to keep the wound environment moist and to absorb exudates. 
	NOTE: The non-adhesive dressings will be removed by the ZDF rats within a few days after application. Therefore, it is necessary to regularly monitor the rats after transplantation. Usually, the non-adhesive dressing is replaced every 2 days under general anesthesia.</li>
	<li>Return each rat that has undergone surgery to a separate cage until they have fully recovered (one rat per cage). After an observation period, euthanize the rats using the approved method of isoflurane overdose.</li>
</ol>

<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>

以下作者披露与此发布相关的财务关系: 辉冈野是创始人和董事的细胞种子有限公司许可证技术和专利从东京女子医科大学，并照雄冈野和雅之大和利益相关者在细胞种子公司东京女子医科大学接收从细胞种子公司研究基金其他作者宣称，他们并没有到此出版物有关的财务关系。

成功地培养区域审计事务中心表最关键的步骤如下: 1) 温度必须在温度反应性培养皿培养过程维持在大约 37 ° C。在区域审计事务中心表的创建，过程中每道工序进行 37 ° C 的热板，和每个试剂被加热到 37 ° C，以防止细胞自发地分离从菜31。2) 收件人 ZDF 大鼠必须进行监测，以防止被删除的非胶粘剂的酱汁，是区域审计事务中心床单移植成功的关键。如果删除了更衣室，必须应用...

糖尿病患者的人口世界范围内增加，达到 4 亿 2015年1;估计有 15-25%的糖尿病患者处于危险从下肢糖尿病溃疡2的进展。糖尿病下肢溃疡是棘手的可能需要长期治疗与康复训练后完全恢复。长治疗期间经常导致显著降低患者的生活质量。因此，治疗糖尿病创面必须开发新的治疗方法，减少或防止恶化。评价糖尿病创面愈合，我们优化糖尿病溃疡创面愈合大鼠模型，模仿实际临床情况，并评估是否移植脂肪源性干细胞 (ASC) 床单使用单元格表工程加速伤口愈合。
骨髓间质细胞 (MSCs) 表现出极大的潜力，加速创面愈合其自我更新的能力，他们的免疫调节作用，它们能够分化成多种细胞谱系3。陶瓷是一种类型的 MSC 来源于脂肪组织，和他们表现出骨髓间充质干细胞来自其他组织，包括其潜在的血管生成和旁分泌活动4，5的几个优势。脂肪组织是较为丰富的人体，并其辅助功能允许收集使用微创手术。因此，陶瓷已用于实验伤口愈合应用6，7。
以前的报道表明，单个单元格 MSC 悬浮液直接注入伤口周围地区可以加快伤口愈合8，9。然而，尽管报告的加速伤口愈合的糖尿病溃疡模型的单细胞悬液注射后，在伤口部位移植细胞的存活时间尚不清楚。
在此研究中，我们应用使用温度反应性培养皿的细胞板工程。这些菜有温度响应聚合物N-异丙基丙烯共价键到10他们表面上。接枝的聚合物层允许温度控制的细胞粘附或脱离表面的培养皿。这道菜的表面变得疏水在 37 ° C，让细胞粘附和增殖，而细胞自发地从表面分离的时候就亲水在温度低于 32 ° c。体外培养的细胞可以收获作为完整细胞到细胞连接与细胞外基质 (ECMs) 的连续单元格表只是通过降低的温度;因此，蛋白水解酶，破坏 ECM，如胰蛋白酶，并非需要的11。因此，细胞板工程可以保护细胞间连接和改善细胞移植的疗效。
此外，细胞板移植增加细胞存活率时相比，细胞注射12。在此协议中，作为延迟的伤口愈合 2 型糖尿病与肥胖模型选取 Zucker 糖尿病脂肪肝 (ZDF) 鼠。在大约 4 个星期中 ZDF 大鼠自发形成肥胖。他们然后患 2 型糖尿病与肥胖之间 8 和 12 周龄，此时他们表现出高血糖与胰岛素抵抗、 血脂异常和甘油13相关联。延迟的伤口愈合，血流量减少外围及血管中，糖尿病肾病也观察到14，，1516。此外，ZDF 大鼠可能适当模型研究顽固性皮肤溃疡，例如糖尿病溃疡的愈合。
人类和啮齿类动物在创面愈合的机制上的差异是与皮肤的解剖学差异相关联。正常大鼠愈合的伤口基于伤口收缩而创面愈合的人类基于重新上皮和肉芽组织的形成。通常情况下，伤口小夹板固定在啮齿动物模型中使用有助于尽量减少伤口收缩并允许逐步形成的肉芽组织17，虽然非糖尿病大鼠伤口已经几乎完全闭合的收缩。然而，糖尿病创面收缩 ZDF 在大鼠是受损，并且伤口愈合主要发生通过重新上皮和肉芽组织的形成;因此，这一进程是更类似于人类的伤口愈合14。
糖尿病伴骨外露创面后清创术临床上经常遇到。先前的研究审查了直径为 12 毫米全层皮肤伤口的裸鼠裸鼠18，19背上和背上的正常小鼠20直径为 10 毫米全层皮肤伤口。发展为严重的糖尿病创面的临床模型，较大的 (15 x 10 mm2) 全层皮肤缺损暴露骨并没有骨膜被创造了，作为先前描述21，大鼠 2 型糖尿病和肥胖。
通过同种异体移植的 ASC 床单创造了大鼠 ASC （区域审计事务中心） 床单从正常 Lewis 大鼠的陶瓷。在临床实践中，自体移植是行不通的因为溃疡的糖尿病患者常表现出严重的糖尿病并发症，如不加控制高血糖和高身体质量指数，这些并发症原因伤口愈合障碍，增加了从这些患者获得脂肪组织的难度。此外，从动物与糖尿病展览体干改变属性和受损功能22。因此，议定书 》 在这里提出了一种描述区域审计事务中心床单从正常大鼠同种异体移植和人工皮肤对糖尿病大鼠的应用。
在本协议中使用的双层人工皮肤防止伤口的自发性收缩、 促进合成的一种新的结缔组织矩阵，和类似于真正的真皮23。在此协议中，人工皮肤是放置区域审计事务中心工作表上，用尼龙线，防止伤口收缩或扩大造成的松散大鼠皮肤固定。此外，人造皮肤提供一种三维框架为 ASC 床单、 保持潮湿的环境下的移植的 ASC 床单和伤口，和保护伤口免受感染和外部力量。最后，非胶粘剂敷料被放置在伤口保护它免受外部冲击、 保持潮湿的伤口环境，和吸收渗出物。
一个区域审计事务中心表是薄薄的、 灵活的和可变形和可以坚持移动收件人的网站，例如24跳动的心。单元格表工程已被用于各种组织重建和可以生成疗效25，26.表现出临床治疗潜力的 ASC 床单可能加速许多类型的伤口的愈合。此外，ASC 床单，结合人工皮肤，使用同种异体移植可能适用于治疗难治性溃疡或烧伤，例如那些观察外周动脉疾病或胶原病，或者他们可能注射到患者营养不良或使用类固醇。这种方法提高移栽体干的效率。伤口愈合 ZDF 大鼠模型产生严重的伤口情况，类似于人类的伤口愈合过程，模仿在小型实验动物临床条件。

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="21">
			<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

人工皮肤取得了相当大的治疗效果，在临床实践中。然而，可能会延长人工皮肤治疗糖尿病患者与阻碍的血流或大的伤口的伤口。作为一种新技术治疗糖尿病溃疡，出现了基于细胞疗法及细胞板工程已改善细胞移植的疗效。大量的报告建议脂肪源性干细胞 （陶瓷），一种间充质基质细胞 (MSC)，表现出由于他们在脂肪组织和其可访问性集合时相比骨髓间充质干细胞从其他组织中的相对丰度的治疗潜力。因此，陶瓷似乎是一个良好的来源的干细胞用于治疗目的。在这项研究，ASC 床单从正常 Lewis 大鼠附睾脂肪已成功创建使用温度反应性培养皿和正常培养基含有抗坏血酸。ASC 床单被移植进 Zucker 糖尿病脂肪酸 (ZDF) 大鼠，大鼠模型的 2 型糖尿病、 肥胖等，表现出减少创面愈合。伤口被创建后颅骨表面、 ASC 床单被移植到伤口，和双层人工皮肤被用来支付床单。收到 ASC 床单的 ZDF 大鼠有更好地愈合比没有移植的 ASC 床单 ZDF 老鼠。这种方法是有限的因为 ASC 床单是敏感的干燥条件，需要一个潮湿的伤口环境的维护。因此，人工皮肤用于封面 ASC，防止干燥。同种异体移植的 ASC 床单结合人工皮肤也可能适用于其他难治性溃疡或烧伤，例如那些观察外周动脉疾病与胶原病和可能给予营养不良或使用类固醇的患者。因此，这种治疗可能改善糖尿病创面愈合的治疗选项的第一步。

本议定书试图建立一种新的基于单元格的难治性糖尿病伤口疗法。简要 （如图 1所示），同种异体区域审计事务中心床单从正常大鼠使用单元格表工程被创造了，然后移植对糖尿病大鼠使用双层人工皮肤全层皮肤缺损。光镜图像的区域审计事务中心表 (图 2A) 的一个好例子和一个坏的榜样的区域审计事务中心表 (图 2B) 如图 2</stron...

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

脂肪干细胞 （陶瓷） 容易孤立和收获从正常大鼠脂肪。ASC 床单可以使用单元格表工程创建，可以移植到 Zucker 糖尿病脂肪肝大鼠表现出全层皮肤缺损伴骨外露，然后用双层的人工皮肤覆盖。

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

creation-transplantation-an-adipose-derived-stem-cell-asc-sheet

Research

JoVE Journal

Medicine

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

11.3K 次观看.  Tokyo Women's Medical University School of Medicine. 脂肪干细胞 （陶瓷） 容易孤立和收获从正常大鼠脂肪。ASC 床单可以使用单元格表工程创建，可以移植到 Zucker 糖尿病脂肪肝大鼠表现出全层皮肤缺损伴骨外露，然后用双层的人工皮肤覆盖。

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

主动脉原位移植大鼠模型，研究慢性血管病变的发展

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

科研

医学

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

一个小鼠模型在子宫内移植

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.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

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.

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.

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

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

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

选择性化学腰椎交感神经切除术中灰色拉米关节的靶向性

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...