Yazarlar Dr Yukiko Koga bölümü plastik ve Rekonstrüktif cerrahi, Juntendo Üniversitesi Tıp Fakültesi, pratik tavsiyeler için teşekkür ederiz. Biz de Bay Hidekazu Murata diyabetik Merkezi, Tokyo kadın Tıp Üniversitesi Tıp Fakültesi mükemmel teknik destek için teşekkür ederiz. Bu çalışmada oluşturma yenilik merkezleri tarafından gelişmekte olan yenilik sistemleri "hücre sayfası doku Mühendisliği Merkezi (CSTEC)" için projenin disiplinler arası araştırma alanları programı gelişmiş için Milli Eğitim Bakanlığı, kültür, spor, bilim ve Teknoloji (MEXT) Japonya'nın desteklenmiştir.

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>

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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>

Finansal ilişkileri bu yayın için ilgili aşağıdaki yazarlar ifşa: Teruo Okano kurucusu ve Yöneticisi, teknoloji ve patent Tokyo kadın Tıp Üniversitesi'nden lisans verir, hücre tohum Inc.'in yönetim kurulu ve Teruo Okano ve Masayuki Yamato paydaşların hücre tohum A.ş Tokyo kadın Tıp Üniversitesi Araştırma Fonu hücre tohum Inc alır Diğer yazarlar onlar finansal ilişkileri bu yayına ilgili yok bildirin.

Bir rASC sayfası başarıyla kültür için en kritik adımlar şunlardır: 1) sıcaklık sıcaklık duyarlı kültür yemekleri kültür sırasında yaklaşık olarak 37 ° C'de saklanması gerekir. Bir rASC sayfası oluşturma sırasında her yordam 37 ° C termo-plaka üzerinde gerçekleştirilmiş ve her reaktif kendiliğinden çanak31ayırma hücreleri önlemek için 37 ° c ısıttı. 2) alıcı ZDF fareler rASC sayfaları başarılı ekimi için kritik olmayan yapışkan soyunma kaldırıl...

Diyabetik hastalar nüfusu dünya çapında artan ve 20151400 milyon ulaştı; Diyabetli hastaların yaklaşık 15-%25 etkilenir alt ekstremite diyabetik ülser2ilerleme. Alt ekstremite diyabetik ülser zorlu ve uzun süreli tedavi nokta ile rehabilitasyon eğitim tam kurtarma sonra gereksinim duyabilir. Uzun tedavi süresi genellikle hastanın yaşam kalitesini önemli bir azalma olur. Böylece, yeni tedaviler tırmandığı önlemek veya azaltmak diyabetik yaralar tedavisi için geliştirilmelidir. Diyabetik yara iyileşmesi değerlendirmek için biz pratik klinik koşulları taklit eder, bir diyabetik ülser yara iyileşmesi modeli içinde rats, en iyi duruma getirilmiş ve değerlendirilmiş olup yağ kaynaklı kök hücre (ASC) yaprak hücre sayfalık mühendislik kullanarak dikim hızlandırılmış yaranın normal iyileşmesi.
Mezenkimal stromal hücre (MSCs) kendi kendini yenileme kapasitesi, immunomodulatory etkileri ve yeteneklerini çeşitli hücre soy3içine ayırmak için nedeniyle yara iyileşmesi hızlandırılması için mükemmel bir potansiyel sergi. ASCs MSC Yağ dokusundan elde edilen bir türüdür ve onların anjiogenik potansiyel ve parakrin etkinlik4,5de dahil olmak üzere diğer dokulardan türetilmiş MSCs çeşitli avantajlar sergilemek. Yağ dokusu insan vücudunda nispeten bol ve minimal invaziv yordamları kullanarak koleksiyonu için erişilebilirlik sağlar. Bu nedenle, ASCs deneysel olarak yara iyileşmesi uygulamaları6,7için kullanılmaktadır.
Önceki raporları tek hücreli MSC süspansiyonlar direkt enjeksiyon yara çevresinde alanlara8,9şifa yara hızlandırabilir göstermiştir. Ancak, tek hücreli süspansiyonlar enjeksiyon takip diyabetik ülser modellerinde yara iyileşmesi ivme raporları rağmen yara yerinde transplante hücre sağkalım süresi net değildir.
Bu çalışmada, biz sıcaklık duyarlı kültür yemekleri kullanarak hücre sayfalık mühendislik uygulanır. Bu yemekleri onların yüzey10kovalent bağlı sıcaklık duyarlı polimer N- isopropylacrylamide var. Aşılı polimer tabaka ısı kontrollü hücre adezyon veya kültür çanak yüzeyinden dekolmanı için izin verir. Çanak yüzeyi 32 ° c altındaki sıcaklıklarda Hidrofil hale geldiğinde hücreleri kendiliğinden yüzeyinden ayırmak ise uygun ve çoğalırlar, hücrelere izin 37 ° C'de hidrofobik hale gelir Kültürlü hücreler bitişik hücre sabıkası olduğu gibi hücre hücre kavşaklar ve ekstraselüler matrisler (ECMs) olarak sadece sıcaklık azaltarak hasat edilebilir; Böylece, ECM, tripsin gibi zarar proteolitik enzimler gerekli11değildir. Bu nedenle, hücre sayfalık mühendislik hücre hücre bağlantıları korumak ve hücre transplantasyonu etkinliğini artırmak.
Ayrıca, hücre-saç ekimi ne zaman hücre enjeksiyon12' ye göre hücre sağkalım oranları artar. Bu protokol için Zucker diyabetik yağlı (ZDF) fareler gecikmiş yara iyileşmesi ile tip 2 diyabet ve obezite model olarak seçildi. ZDF fareler kendiliğinden yaklaşık 4 hafta obezite geliştirmek. Daha sonra 8 ile 12 hafta-in yaş, bu noktada onlar insülin direnci, dislipidemi ve hipertriglisemik13ile ilişkili hiperglisemi sergi arasında obezite ile tip 2 diyabet geliştirmek. Gecikmiş yara periferik kan damarları ve diyabetik nefropati azaltılmış kan akımını şifa, ayrıca14,15,16görülmektedir. Ayrıca, ZDF fareler zorlu Kutanöz ülser, diyabetik ülser gibi şifa eğitim için uygun bir model olabilir.
Yara iyileşmesi mekanizmaları kemirgenler ve insanlar arasındaki farklılıkları deride anatomik farklılıklar ile ilişkilidir. İse insanlarda yara iyileşmesi yeniden epithelialization ve granülasyon dokusu oluşumu üzerinde de, normal fareler şifa yara yara kasılması, temel alır. Genellikle, kemirgen modellerinde kullanılan yara splint yara daralma en aza indirmek için yardımcı olur ve yaralar diyabetik Sıçanlarda neredeyse tamamen daralma tarafından kapatılır, ancak granülasyon dokusu17, kademeli oluşumu için sağlar. Ancak, daralma diyabetik yara ZDF içinde fareler Engelli ve öncelikle şifa yara oluşur yeniden epithelialization ve granülasyon dokusu oluşumu; Bu nedenle, bu işlem daha14şifa insan yara benzerdir.
Debridman sık karşılaşılan sonra klinik olarak maruz kalan kemik ile diyabetik yaralar. Önceki çalışmalarda 12 mm çap tam kalınlıkta deri yaraları korele çıplak fareler18,19 sırtında ve 10 mm çap tam kalınlıkta deri yaraları normal farelere20sırtında inceledik. Şiddetli diyabetik yaralar için klinik bir model geliştirmek için daha büyük (15 x 10 mm2) tam kalınlıkta deri kusurları ile kemik maruz ve kapaklarini olarak yukarıda açıklanan21, tip 2 diyabet ve obezite ile Sıçanlarda oluşturulan.
Sıçan ASC (rASC) normal Lewis sıçan ASCs sayfalarından ASC sayfaları allojenik nakli oluşturulmuştur. Diyabetik hastalarda ülser kez kontrolsüz yüksek kan şekeri ve yüksek vücut kitle endeksi ve yağ dokusu bu hastalardan elde etme zorluğu artırmak bu komplikasyonlar neden yara iyileşmesi bozukluklar gibi ağır diyabetik komplikasyonlar sergilemek çünkü klinik uygulamada, Otolog transplantasyon olanaksız. Ayrıca, ASCs diyabet sergi ile hayvanlardan özellikleri değişmiş ve işlev22Engelli. Bu nedenle, normal sıçanlar rASC sayfalarından allojenik nakli ve Yapay cilt uygulamaya diyabetik sıçan burada sunulan protokolünü açıklar.
Bu protokol için kullanılan bilayer Yapay deri yaraları spontan daralma engeller, yeni bir bağ dokusu matris sentezini teşvik ve gerçek DermIS23benzer. Bu protokol için yapay deri bir rASC sayfaya yerleştirilen ve yara daralma veya gevşek sıçan derisinden kaynaklanan genişleme önlemek için naylon konu ile sabit. Buna ek olarak, suni deri için ASC yaprak üç boyutlu bir çerçeve sağlar, transplante ASC sayfaları ve yaralar için nemli bir ortamın yaratıldığı ve yaralar enfeksiyon ve dış güçler korur. Son olarak, yapışkan olmayan soyunma dış etki korumak, nemli yara ortamı korumak ve çıktı absorbe yara yerleştirilir.
Bir rASC sac ince, esnek ve deforme ve dayak kalp24gibi alıcı siteler hareket için yapıştırılır. Hücre sayfalık mühendislik çeşitli dokuların yeniden inşası için kullanılan ve iyileştirici etkileri25,26. -ebilmek oluşturmak Klinik tedavi potansiyel sergi ASC sayfaları yaralar birçok türleri konusunda bilgi sahibi şifa hızlandırmak. Ayrıca, allojeneik nakli ASC yaprak, suni deri, kullanımı ile kombine tedaviye dirençli ülser veya burns, bu periferik arter hastalığı veya kollajen hastalığı, gözlenen gibi geçerli olabilir veya Beslenme yetersizliği veya steroid kullanan hastalar için yönetilen. Bu yaklaşım ASCs nakli verimliliğini artırır. Yara iyileşmesi ZDF fare modeli işlem şifa insan yara benzer ve küçük ölçekli deneysel hayvan klinik koşullarda taklit eden bir ağır yara koşul üretir.

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			<td height="21" style="height:21px;width:223px;">&#945;-MEM glutamax</td>
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			<td style="width:167px;">Carlsbad, CA</td>
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			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Japan Bioserum Co Ltd.</td>
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			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Life Technologies</td>
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			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche Diagnostics</td>
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			<td height="25" style="height:25px;">60-cm2 Primaria tissue culture dish</td>
			<td>BD Biosciences</td>
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			<td>Franklin Lakes, NJ</td>
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			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate Buffer Saline (PBS)</td>
			<td>Life Technologies</td>
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			<td height="21" style="height:21px;">0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td>
			<td>Life Technologies</td>
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			<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>
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			<td height="25" style="height:25px;">35-mm temperature-responsive culture dish (UpcellTM)</td>
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			<td height="42" style="height:42px;">Microwarm plate (MP-1000)</td>
			<td style="width:169px;">Kitazato Science Co., Ltd.</td>
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			<td height="21" style="height:21px;">4% isoflurane</td>
			<td>Pfizer Japan</td>
			<td>114-13340-3</td>
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			<td height="25" style="height:25px;">Artificial skin (Pelnac&#174;)</td>
			<td>Smith &#38; Nephew</td>
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			<td>&#160;Tokyo, Japan</td>
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			<td height="25" style="height:25px;">Non-adhesive dressing (Hydrosite plus&#174;)</td>
			<td>Smith &#38; Nephew</td>
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			<td>Known as Allevyn non-adhessing&#174; in the United State</td>
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			<td>Alfresa</td>
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			<td>greiner bio-one</td>
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			<td height="21" style="height:21px;width:223px;">15mL Centrifuge Tube</td>
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			<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>
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creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model

Yapay cilt klinik pratikte önemli tedavi edici sonuçlar elde etti. Ancak, Yapay cilt tedavileri engellemiştir kan akımı veya büyük yaralar ile diyabetik hastalarda yaralar için uzun süreli. Hücre tabanlı terapiler diyabetik ülser tedavisi için yeni bir teknik olarak yer aldı ve hücre sayfalık mühendislik hücre transplantasyonu etkinliğini geliştirdi. Raporlar bir dizi yağ kaynaklı kök hücreler (ASCs), bir tür Mezenkimal stromal hücre (MSC), yağ dokusu ve onların erişilebilirlik için MSCs diğer dokulardan karşılaştırıldığında koleksiyonu için göreli onların bereket nedeniyle tedavi potansiyel sergi tavsiye ettiler. Bu nedenle, ASCs kök hücre terapötik kullanım için iyi bir kaynak gibi görünüyor. Bu çalışmada, ASC sayfaları normal Lewis sıçan epididimal yağ yağ sıcaklık duyarlı kültür yemekleri ve normal kültür askorbik asit içeren orta kullanarak başarıyla oluşturuldu. ASC sayfaları Zucker diyabetik yağ (ZDF) fareler nakledilen, yaranın normal tip 2 diyabet ve obezite, azalmış sorun oluşturan bir fare modeli. Bir yara arka kafatası yüzeyinde oluşturuldu, ASC sayfaları yara içine nakledilen ve bilayer Yapay deri çarşafları kapsayacak şekilde kullanıldı. ASC sayfaları alınan ZDF fareler daha iyi ZDF fareler ASC sayfaları nakli olmadan daha yaranın. ASC sayfası nemli yara çevre bakım gerektiren kuru şartlarda için duyarlı olduğundan bu yaklaşım sınırlıydı. Bu nedenle, Yapay cilt kurutma önlemek için ASC sayfası kapsayacak şekilde kullanıldı. ASC sayfaları Yapay deri ile birlikte allojenik nakli de diğer zorlu ülser veya burns, bu kollajen hastalığı, periferik arter hastalığı ile gözlenen gibi geçerli olabilir ve Beslenme yetersizliği veya steroid kullanan hastalar için yönetilen. Böylece, bu tedavi diyabetik yara iyileşmesi için tedavi olanakları geliştirmeye yönelik ilk adım olabilir.

Bu iletişim kuralı bir yeni hücre tabanlı terapi dirençli diyabetik yaralar için kurmak çalıştı. (Olarak şekil 1' deki resimli), kısaca allojeneik rASC sayfaları normal sıçanlarından emir hücre sayfalık mühendislik kullanılarak oluşturulmuş ve sonra suni deri üzerine bir tam kalınlıkta deri kusur bilayer bir diyabetik sıçan kullanarak nakledilmektedir. İyi bir örnek bir rASC sayfasının (şekil 2A) ve kötü bir örnek bir rA...

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

Yağ kaynaklı kök hücreler (ASCs) kolayca izole ve normal sıçanlar FAT'tan hasat. ASC yaprak hücre sayfalık mühendislik kullanılarak oluşturulabilir ve tam-kalınlık sergilenmesi diyabetik yağlı sıçanlar kusurları maruz kemik ile deri ve suni deri bilayer ile kaplı Zucker içine nakledilmektedir.

Oluşturma ve diyabetik yara iyileşmesi manken bir yağ elde edilen kök hücre (ASC) Sheet nakli

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 Görüntüleme.  Tokyo Women's Medical University School of Medicine. Yağ kaynaklı kök hücreler (ASCs) kolayca izole ve normal sıçanlar FAT'tan hasat. ASC yaprak hücre sayfalık mühendislik kullanılarak oluşturulabilir ve tam-kalınlık sergilenmesi diyabetik yağlı sıçanlar kusurları maruz kemik ile deri ve suni deri bilayer ile kaplı Zucker içine nakledilmektedir.

Video: 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.

Ortotopik Aort Nakli: Rat Modeli Kronik Vaskülopati Geliştirme Çalışması

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

Tıp

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

Bir Fare Modeli Utero Organ Nakli

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.

Bir Kök Hücre Nakli In vitro Simüle İskemi / Reperfüzyon Modeli

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.

Rekabetçi bir mürin Kemik İliği Transplantasyon Modelinde Donör Hücre Aşı belirleme Kantitatif Gerçek-zamanlı PCR kullanarak

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.

Yara Şifa murin Modeli

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.

Bir sıçan modelinde kısmi kolektomi sonra kaçağı azaltmak için Yağ dokusundan elde edilen kök hücre sayfasının nakli

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.

Kornea epitel aşınma oküler Burr ile bir Model olarak kornea için yara iyileşme

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.

Ex vivo kornea Organ kültür modeli için yaranın çalışmaları

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.

Seçmeli kimyasal Lomber sempatektomi gri Rami Communicantes hedefleme

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.

Bir Ex Vivo doku kültürü modeli Fibrovascular komplikasyon proliferatif diyabetik retinopati

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