Name: creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model
Uploaded: 2017-08-04T12:30:00.000+00:00
Duration: 8 min 6 s
Description: Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

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>

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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><tbody><tr><td>&amp;alpha;-MEM glutamax</td><td>Invitrogen</td><td>32571-036</td><td>Carlsbad, CA</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Japan Bioserum Co Ltd.</td><td>S1650-500</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Collagenase A</td><td>Roche Diagnostics</td><td>10 103 578 001</td><td>Mannheim, Germany</td></tr><tr><td>60 cm&lt;sup&gt;2&lt;/sup&gt; Primaria tissue culture dish</td><td>BD Biosciences</td><td>353803</td><td>Franklin Lakes, NJ</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffer Saline (PBS)</td><td>Life Technologies</td><td>1490-144</td><td></td></tr><tr><td>0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>L-ascorbic acid phosphate magnesium salt n-hydrate</td><td>Wako</td><td>013-19641</td><td></td></tr><tr><td>35-mm temperature-responsive culture dish (Upcell&lt;sup&gt;TM&lt;/sup&gt;)</td><td>CellSeed</td><td>NUNC-174904</td><td>Tokyo, Japan</td></tr><tr><td>Microwarm plate (MP-1000)</td><td>Kitazato Science Co., Ltd.</td><td>1111</td><td></td></tr><tr><td>Rodent mechanical ventilator</td><td>Stoelting</td><td>#50206</td><td>Wood Dale, IL</td></tr><tr><td>4% isoflurane</td><td>Pfizer Japan</td><td>114-13340-3</td><td>Tokyo, Japan</td></tr><tr><td>Artificial skin (Pelnac&lt;sup&gt;&amp;reg;&lt;/sup&gt;)</td><td>Smith &amp;amp; Nephew</td><td>PN-R40060</td><td>&amp;nbsp;Tokyo, Japan</td></tr><tr><td>Non-adhesive dressing (Hydrosite plus&lt;sup&gt;&amp;reg;&lt;/sup&gt;)</td><td>Smith &amp;amp; Nephew</td><td>66800679</td><td>Known as Allevyn non-adhessing&lt;sup&gt;&amp;reg;&lt;/sup&gt; in the United State</td></tr><tr><td>5-0 nylon suture</td><td>Alfresa</td><td>EP1105NB45-KF2</td><td></td></tr><tr><td>20 CELLSTAR TUBES</td><td>greiner bio-one</td><td>227 261</td><td></td></tr><tr><td>15 mL Centrifuge Tube</td><td>Corning Incorporated</td><td>430791</td><td></td></tr><tr><td>14 GOLDMAN-FOX PERIOSTEAL</td><td>Hu-Friedy</td><td>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

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

一个生产缺血性损伤模型的简化技术

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

科研

医学

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

生物工程

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

人体外伤模型和全山染色方法，以准确评估皮肤修复

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

发育生物学

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

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

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

ADSC表移植到扩展食管内镜黏膜下剥离术之后防止狭窄

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

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 ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

在糖尿病小鼠中创建慢性伤口的协议

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with dressing changes, hyperbaric oxygen, and vacuum sealing drainage are the main conservative treatments in clinical practice, and large wounds often require skin grafts or skin flap grafts. However, the treatment effects are not ideal, and many complications exist. Due to its complex pathogenesis, long treatment time, significant associated difficulties, and high disability rate, diabetic foot ulcers cause a heavy burden to patients, society, and medical care. According to our previous study, the pharmacological effects of human umbilical cord blood stem cells include nonspecific immune regulation; increased secretion of growth factors, vasoactive factors, and anti-inflammatory factors; enhanced anti-infectious ability of the human body; elimination of inflammation; and promotion of angiogenesis and ulcer healing. These effects suggest stem cells may be useful as an autologous or allogeneic treatment for refractory wounds. Therefore, we are conducting a clinical trial to treat refractory diabetic wounds with human umbilical cord stem cells in our clinic for diabetic foot ulcer patients who meet the inclusion criteria.

The present protocol describes a prospective, randomized, controlled clinical study that evaluates a human umbilical cord mesenchymal stem cell injection for the treatment of chronic diabetic foot ulcers.

人脐带间充质干细胞注射治疗糖尿病足溃疡的前瞻性、随机和对照研究

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

毛里岛皮下白脂肪组织（ISWAT）移植模型

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

使用核心鞘 3D 生物打印支架优化细胞外囊泡递送用于慢性伤口管理

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

一种构建1型糖尿病大鼠伤口模型的方案

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...