作者感谢 S.E.R. Hovius 博士, M.A.M. 博士 Mureau 和整形外科部的所有外科医生, 以收集皮下脂肪组织。p. Sukho 得到荷兰研究金计划 (NFP-12/435) 的赠款支持, 在进行这项学习期间。Y.M. Bastiaansen-Jenniskens 由荷兰关节炎基金会 (LP11) 提供资助。

皮下腹部脂肪组织是由人类捐赠者获得的医学伦理委员会 (#MEC 2014-092), 荷兰鹿特丹的伊拉斯谟 MC 大学医疗中心批准。所有动物实验均经动物实验伦理委员会批准, 荷兰鹿特丹伊拉大学医学中心 (133-14-01)。
1. 人类陶瓷隔离和文化
<ol><li>为1克脂肪组织准备3毫升的隔离培养基。混合低葡萄糖 Dulbecco 的改良鹰的培养基 (LG-DMEM) 与10克/升牛血清白蛋白 (BSA) 和1克/升胶原酶1型。</li>
	<li>解剖人皮下腹部脂肪组织 (n = 8, 所有女性, 平均年龄 40 9 岁) 到小块 (0.5 厘米) 使用无菌手术刀片大小 10, Adson 棕色组织钳和 Metzenbaum 剪刀在一个无菌的生物 SafetyCabinet。</li>
	<li>将解剖过的组织保存在无菌玻璃培养基储存瓶中。用准备好的隔离培养基 (每瓶50克脂肪组织150毫升的隔离培养基) 来衡量解剖组织的总数量, 开始消化。该协议可以暂停在这里通过储存与解剖脂肪组织和隔离介质在一个滚筒搅拌机4°c 过夜。然后 prewarm 瓶第二天的室温25°c 为15分钟, 然后继续下一步。 注: 皮下腹部脂肪组织是由捐赠者进行乳房重建手术的剩余材料获得的, 并经伊拉兹 MC 医学伦理委员会 (MEC-200) 批准, 并根据行为守则: "适当的二次使用人的组织 "(< http://www.federa.org >)。剩下的脂肪组织只用于不选择二次使用的捐赠者。</li>
	<li>消化在一个无菌玻璃培养基储存瓶中的解剖脂肪组织在 150 rpm 和37°c 1 小时的振动筛孵化器。</li>
	<li>将所消化溶液分为50毫升管, 离心管10分钟 390 x 克。</li>
	<li>制备培养基: LG-DMEM 辅以10% 胎牛血清 (血清), 50 µg/毫升庆大霉素, 1.5 µg/毫升 ampothericin B。</li>
	<li>离心后, 去除上清和并用重悬细胞颗粒在20毫升培养基 (LG-DMEM 补充10% 胎牛血清 (血清), 50 µg/毫升庆大霉素和1.5 µg/毫升 ampothericin B)。再次离心细胞5分钟在 390 x g 和删除上清。</li>
	<li>并用重悬细胞颗粒与10毫升培养基 (LG-DMEM 补充10% 血清, 50 µg/毫升庆大霉素和1.5 µg/毫升 ampothericin B) 和过滤细胞悬浮通过100µm 过滤器。</li>
	<li>添加50µL 3% 醋酸/亚甲基蓝溶液 (用于红细胞裂解) 到50µL 细胞悬浮液, 混合溶液并使用10µL 计数细胞。使用 hemocytometer 执行单元格计数。然后板细胞在密度4万细胞/cm2在培养基 (LG-DMEM 补充30% 血清, 50 µg/毫升庆大霉素和1.5 µg/毫升 ampothericin B)。</li>
	<li>在潮湿的大气层中孵化出37摄氏度的细胞, 5% CO2为24小时。</li>
	<li>孵化后, 用温 (37 °c) 磷酸盐缓冲生理盐水 (PBS) 冲洗细胞, 去除细胞碎片, 用新鲜添加的抗坏血酸 acid-2-phosphate (25 µg/毫升) 和人重组成纤维细胞生长的10% 只血清培养基取代培养基。因子 2 (FGF2, 1 ng/毫升)。</li>
	<li>亚文化陶瓷在90% 汇合使用标准0.25% 胰蛋白酶 EDTA 溶液 (3 毫升为 T175 瓶)。3-5 分钟后, 中和0.25% 胰蛋白酶的 EDTA 溶液与10毫升培养基 (LG-DMEM 补充10% 血清, 50 µg/毫升庆大霉素和1.5 µg/毫升 ampothericin B)。 注: 使用常见的 ASC 表面标记和三谱系分化检测的流式细胞术分析表明, 陶瓷隔离使用此协议显示 ASC 特定特性21,22。</li>
	<li>用10毫升培养基 (LG-DMEM 补充10% 的血清) 冲洗细胞, 50 µg/毫升庆大霉素和1.5 µg/毫升 ampothericin B) 为 T175 瓶和离心机的细胞为8分钟在 250 x g. 移除上清, 并在1毫升培养基中并用重悬细胞。用 hemocytometer 计数单元格。</li>
	<li>T175 培养皿中的细胞以8000细胞/厘米2的密度为单位。</li>
	<li>在进一步使用前, 在培养基中用10% 二甲基亚砜 (亚砜) 冻结液氮中剩余的陶瓷。</li>
</ol>2. ASC 板材的制备
<ol><li>预涂层热响应培养皿 (3.5 厘米直径) 与1毫升的血清, 然后将菜肴放在一个孵化器37°c 至少30分钟之前, 细胞播种。</li>
	<li>胰蛋白酶处理陶瓷使用标准的0.25% 胰蛋白酶 EDTA 溶液3-5 分钟 (3 毫升的 T175 培养瓶)。如果 trypsinization 后有任何细胞团簇, 请在计数前通过100µm 过滤器过滤细胞。</li>
	<li>中和的胰蛋白酶溶液与 LG-DMEM 补充10% 的血清 (10 毫升的 T175 瓶) 和离心机的细胞悬浮8分钟在 250 x g. 去除上清和并用重悬的细胞在1毫升 LG-DMEM 补充10% 血清。</li>
	<li>涂上热响应的文化菜肴与血清, 移动从孵化器的菜肴, 以37摄氏度的暖板, 并从盘子中删除的血清。</li>
	<li>种子陶瓷在40万个细胞/cm2 (总, 3.52 x 106细胞稀释在2毫升培养基每菜)。</li>
	<li>通过轻轻摆动盘子, 尽可能均匀地分配细胞。</li>
	<li>文化 ASC 板材为48小时在37°c 在潮湿的大气与 5% CO2。 注: 尽量减少在播种陶瓷后打开孵化器的门, 以防止可能干扰 ASC 板形成或导致形成的 asc 板过早脱离的温度下降。</li>
</ol>3. 部分结肠切除和结直肠吻合术
<ol><li>诱导和维持雄性大鼠 (重量在250-350 克之间) 与1升/分异氟醚 (1.5-5%)/吸入氧和注射0.05 毫克/千克丁丙诺啡肌肉。</li>
	<li>一旦动物全身麻醉, 使用反射测试评估麻醉深度。将含有眼膏的维生素 a 应用于眼部, 以防止干燥。当麻醉被认为是足够的手术, 灌装准备腹部剃须毛和喷洒手术面积两次与70% 乙醇。用不育的纸帘遮住腹部。使用无菌技术, 在整个手术过程中保持无菌场。</li>
	<li>做一个5厘米的中线腹部切口与无菌手术刀片大小10和延伸切口用 Metzenbaum 剪刀。鉴别并 exteriorize 结肠, 用生理盐水湿润的纱布将结肠从腹腔的其余部分包裹起来。确定肠系膜的右、中、左绞痛动脉, 用霍尔斯特德蚊子直接解剖每条血管, 并结扎每条单独的血管, 不吸收编织丝4/0。</li>
	<li>将结肠段从1.0 厘米 aborally 到盲肠, 在肠系膜上动脉的0.5 厘米之间分离。用 Metzenbaum 剪刀通过健康的结肠样带。结肠段切除后, 将两条长的棉拭子通过远端结肠肛门, 然后进入近端结肠末端, 将结肠的近端和远端端一起引入。</li>
	<li>使用手术显微镜, 通过5条间断缝合 (不可吸收的单丝聚酰胺 8/0), 通过一层反转缝合, 建立一个不够缝合的端到端吻合术。将被打断的缝线穿过结肠壁的所有层, 并将结 extraluminally。</li>
	<li>将大鼠随机分成对照组或 ASC 表。</li>
</ol>4. ASC 表移植
<ol><li>在体内移植前, 允许培养皿降温至室温40分钟, 以促进 ASC 板脱离。 注: 在支队后, ASC 床单收缩到约2厘米直径。在支队20之后, ASC 板的生存能力仍然高至少3-6 小时。</li>
	<li>去除培养基, 用1毫升的血清游离 LG DMEM 代替。</li>
	<li>用非创伤性钳轻轻抓住 asc 板的轮辋, 将 asc 片的盘面放在吻合线的顶端。</li>
	<li>仔细伸展的 ASC 表约0.25 厘米以上和下方的吻合线使用非创伤性钳和包装的床单周围的结肠。提起冒号, 将 ASC 片环绕在背侧。 注意: ASC 床单自发地附着在吻合线上, 不需要使用组织胶水或任何其他生物材料。对照组不接受任何额外治疗。</li>
	<li>取出棉签, 更换手套和器械。替换腹部的结肠。用可吸收的编织羟基酸5/0 关闭腹部 linea 和一层连续缝合线。用可吸收的编织羟基酸5/0 将皮下组织和皮肤缝合在一层连续缝合线上。</li>
</ol>5. 术后评价和后续行动
<ol><li>立即水化动物的皮下注射5毫升温盐水术后。将动物置于热灯下, 保持体温, 监测生命体征, 直到动物恢复足够的意识以维持胸骨卧床。 注意: 经过手术的动物直到完全恢复后才返回其他动物公司。恢复后, 允许免费进入水和常规鼠周。手术后止痛, 每6-8 小时管理0.05 毫克/千克丁丙诺啡, 3 天。本研究中任何时候都没有使用抗生素。</li>
	<li>获得健康和体重的每日临床评估。如果有很低的健康评分或严重的体重下降, 动物应该被放血通过心脏穿刺, 而仍然在麻醉下安乐死。</li>
	<li>术后3天或7天, 麻醉大鼠再用1升/分异氟醚 (1.5-5%)/氧吸入无丁丙诺啡, 并进行再剖腹手术的 U 形切口。检查腹部是否有腹膜炎、狭窄、纤维粘连和脓肿的出现。评分在腹部和吻合区的粘连和脓肿的严重性。</li>
	<li>在结肠闭合段包括吻合术中, 确定喷洒的吻合口破裂压力。当第一次空气泄漏为爆破压力时, 记录气压和破裂处。</li>
	<li>从每只牺牲的动物中取出结肠, 固定结肠结直肠吻合的切口段--用4% 缓冲的甲醛, 下面是石蜡嵌入, 将结肠切成4微米厚的部分。</li>
	<li>弄死通过心脏穿刺放血采集吻合段后直接麻醉下的动物。在没有呼吸运动和心跳的情况下确认动物死亡。</li>
	<li>执行组织化学染色, 如苏木精 & 血红素和 Picro 天狼星红, 免疫组化染色抗体抗人线粒体, 大鼠内皮细胞 (anti-CD34) 和细胞 (CD3+, CD163+)。</li>
	<li>数字化电脑分析的幻灯片, 并比较动物组。</li>
</ol>

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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=Adipose-tissue-derived+Stem+Cells+Enhance+the+Healing+of+Ischemic+Colonic+Anastomoses:+An+Experimental+Study+in+Rats.">Adipose-tissue-derived Stem Cells Enhance the Healing of Ischemic Colonic Anastomoses: An Experimental Study in Rats.</a> J Korean Soc Coloproctol. 28 (3), 132-139 (2012).</li><li>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.">Cell sheet engineering.</a> Rinsho Shinkeigaku. 46 (11), 795-798 (2006).</li><li>Chen, G., 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=Application+of+the+cell+sheet+technique+in+tissue+engineering.">Application of the cell sheet technique in tissue engineering.</a> Biomed Rep. 3 (6), 749-757 (2015).</li><li>Labbe, B., Marceau-Fortier, G., Fradette, J. <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+technology+for+tissue+engineering:+the+self-assembly+approach+using+adipose-derived+stromal+cells.">Cell sheet technology for tissue engineering: the self-assembly approach using adipose-derived stromal cells.</a> Methods Mol Biol. 702, 429-441 (2011).</li><li>Sukho, P., 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=Adipose+Tissue-Derived+Stem+Cell+Sheet+Application+for+Tissue+Healing+In+Vivo:+A+Systematic+Review.">Adipose Tissue-Derived Stem Cell Sheet Application for Tissue Healing In Vivo: A Systematic Review.</a> Tissue Eng Part B-Re. , (2017).</li><li>Sukho, P., 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=Effects+of+adipose+stem+cell+sheets+on+colon+anastomotic+leakage+in+an+experimental+model:+Proof+of+principle.">Effects of adipose stem cell sheets on colon anastomotic leakage in an experimental model: Proof of principle.</a> Biomaterials. 140, 69-78 (2017).</li><li>Verseijden, 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=Angiogenic+capacity+of+human+adipose-derived+stromal+cells+during+adipogenic+differentiation:+an+in+vitro+study.">Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation: an in vitro study.</a> Tissue Eng Pt A. 15 (2), 445-452 (2009).</li><li>Sukho, P., 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=Effect+of+Cell+Seeding+Density+and+Inflammatory+Cytokines+on+Adipose+Tissue-Derived+Stem+Cells:+an+in+Vitro+Study.">Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study.</a> Stem Cell Rev. 13 (2), 267-277 (2017).</li><li>. Cell Harvesting by Temperature Reduction Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/brochures/AppNote-Nunc-UpCell-N20230.pdf">https://tools.thermofisher.com/content/sfs/brochures/AppNote-Nunc-UpCell-N20230.pdf</a> (2008)</li><li>Wu, Z., 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=Colorectal+anastomotic+leakage+caused+by+insufficient+suturing+after+partial+colectomy:+a+new+experimental+model.">Colorectal anastomotic leakage caused by insufficient suturing after partial colectomy: a new experimental model.</a> Surg Infect (Larchmt). 15 (6), 733-738 (2014).</li><li>Wu, Z., 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=Reducing+colorectal+anastomotic+leakage+with+tissue+adhesive+in+experimental+inflammatory+bowel+disease.">Reducing colorectal anastomotic leakage with tissue adhesive in experimental inflammatory bowel disease.</a> Inflamm Bowel Dis. 21 (5), 1038-1046 (2015).</li><li>Wu, Z., 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=Reducing+anastomotic+leakage+by+reinforcement+of+colorectal+anastomosis+with+cyanoacrylate+glue.">Reducing anastomotic leakage by reinforcement of colorectal anastomosis with cyanoacrylate glue.</a> Eur Surg Res. 50 (3-4), 255-261 (2013).</li><li>Aysan, E., Dincel, O., Bektas, H., Alkan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polypropylene+mesh+covered+colonic+anastomosis.+Results+of+a+new+anastomosis+technique.">Polypropylene mesh covered colonic anastomosis. Results of a new anastomosis technique.</a> Int J Surg. 6 (3), 224-229 (2008).</li><li>Suarez-Grau, J. 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=Fibrinogen-thrombin+collagen+patch+reinforcement+of+high-risk+colonic+anastomoses+in+rats.">Fibrinogen-thrombin collagen patch reinforcement of high-risk colonic anastomoses in rats.</a> World J Gastrointest Surg. 8 (9), 627-633 (2016).</li><li>Kobayashi, J., 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=Fabrication+of+a+thermoresponsive+cell+culture+dish:+a+key+technology+for+cell+sheet+tissue+engineering.">Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering.</a> Sci Technol Adv Mat. 11 (1), 014111 (2010).</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>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>Makarevich, P. I., 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=Enhanced+angiogenesis+in+ischemic+skeletal+muscle+after+transplantation+of+cell+sheets+from+baculovirus-transduced+adipose-derived+stromal+cells+expressing+VEGF165.">Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165.</a> Stem Cell Res Ther. 6, 204 (2015).</li></ol>

提交人声明, 这项研究是在没有任何商业或金融关系的情况下进行的, 这可能被理解为潜在的利益冲突。

吻合口漏是结肠切除术后最严重的不良事件。目前还缺乏预防吻合口破裂和渗漏的最佳技术。对生物材料阵列的局部应用进行了研究, 结果25、26、27不同。细胞疗法的目的是通过组织置换或通过分泌分泌刺激局部愈合来促进组织修复。
在这个大鼠模型中, ASC 片移植技术上是成功的。临床和组织学评价表明, ...

部分结肠切除与原发性吻合术是一个常见的手术, 可以做许多疾病, 如结肠直肠癌, 克罗恩病和憩1,2。结直肠吻合术后最可怕的并发症是吻合口漏3。虽然已经确定了与吻合口漏相关的几个危险因素, 但防止渗漏的解决方案仍然未知4,5。
脂肪组织衍生基质细胞 (陶瓷) 与抗炎和营养性质的6,7, 使这些细胞有望候选的再生疗法8。陶瓷促进组织愈合的有效性表现在各种组织, 如心肌, 皮肤, 食管9,10,11,12,13。然而, 很少有关于使用陶瓷促进肠道愈合的报道。陶瓷局部移植通过 ASC 包膜 biosutures 或浆膜注射陶瓷的实验性结直肠吻合术14的愈合或没有预防吻合口瘘, 尽管更有利的吻合口愈合15。
局部移植陶瓷悬浮或与生物材料结合可能与细胞保留不足或移植细胞分配不足11。细胞板技术16,17提供了一个创新的方法, ASC 交付18,19。因此, 在以前的研究中, 提出了一种新的方法, 其中陶瓷组织在细胞片可以应用于实验性结直肠吻合20。本研究表明, ASC 片移植成功地减少了大鼠部分结肠切除术后结直肠吻合口渗漏。本文报告了 ASC 板的制备和手术移植技术。

<table border="0" cellpadding="0" cellspacing="0" width="689">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">LG DMEM</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">22320022</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Collagenase type I</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">17100-01</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">A9418</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal bovine serum</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">10270-106</td>
			<td style="width:167px;">FBS, ASC isolation and culture</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">3% acetic acid with methylene blue</td>
			<td style="width:115px;">Stemcell Technologies</td>
			<td style="width:191px;">7060</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Gentamicin</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">15750-037</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ampothericin B&#160;</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">15290-018</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Shaker (Gallenkamp Environmental Shaker Model 10X 400)</td>
			<td style="width:115px;">Akribis Scientific</td>
			<td style="width:191px;">F240</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge&#160;</td>
			<td style="width:115px;">Hettichlab&#160;</td>
			<td style="width:191px;">Rotina380</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:216px;">Phosphate buffer saline&#160;</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">14190-094</td>
			<td style="width:167px;">PBS, ASC isolation and culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ascorbic acid-2-phosphate</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">A8960</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">Human recombinant fibroblast growth factor 2&#160;</td>
			<td style="width:115px;">AbD Serotec</td>
			<td style="width:191px;">AF-100-15</td>
			<td style="width:167px;">FGF2, ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin EDTA</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">25200-056</td>
			<td style="width:167px;">ASC culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">D2650-5x</td>
			<td style="width:167px;">DMSO, ASC freezing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Thermo-responsive culture dishes</td>
			<td style="width:115px;">Nunc Thermo scientific</td>
			<td style="width:191px;">174904</td>
			<td style="width:167px;">ASC sheet preperation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Non-absorbable braided silk 4/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">21151042</td>
			<td style="width:167px;">surgery</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Non-absorbable monofilament polyamide&#160; 8/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">G1118170</td>
			<td style="width:167px;">surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Absorbable braided polyglycolic acid 5/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">C1048207</td>
			<td style="width:167px;">surgery</td>
		</tr>
	</tbody>
</table>

transplantation of adipose tissue-derived stem cell sheet to reduce leakage after partial colectomy in a rat model

吻合口瘘是结直肠手术后的灾难性并发症。虽然目前的防渗漏方法有不同的临床疗效水平, 但目前尚不完善的解决方案。干细胞治疗使用 ASC 表可以提供一个解决这个问题。陶瓷被认为是有希望的候选者, 促进组织愈合, 因为他们的营养和免疫调节性能。在这里, 我们提供的方法, 以生产高密度 ASC 表, 移植到结肠直肠吻合的大鼠模型, 以减少渗漏。陶瓷形成的细胞表在热反应的文化菜肴, 可以很容易地分离。在移植当天, 进行了5缝合结直肠吻合的部分结肠切除术。动物被立即移植与 1 ASC 表每鼠。ASC 床单自发地附着在没有任何胶水, 缝合, 或任何生物材料的吻合。动物群在术后3和7天被献祭。与移植动物相比, 控制动物的吻合口脓肿和渗漏率较高。在本模型中, 结肠吻合术后的 ASC 片移植成功, 并伴有较低的漏率。

图 1显示了这项研究的流程图, 其中描述了 ASC 片培养和结肠切除吻合的过程。图 2显示了 asc 板的显微形貌和在分离期间和之后的 asc 片的宏观外观。图 3显示了 ASC 板剥离和移植的不同步骤。图 4显示了 ASC 表在吻合线上的存在, 并在术后3天内预防渗漏。
<p class="jove_content" fo:keep-toge...

Watch this Scientific Journal Video about Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model at JoVE.com

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

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

对大鼠模型中脂肪组织衍生干细胞 (ASC) 的制备和移植进行了未充分缝合的结直肠吻合。这项新的应用表明, ASC 片可以减少结直肠吻合口漏。

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

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7.5K 次观看.  Utrecht University. 对大鼠模型中脂肪组织衍生干细胞 (ASC) 的制备和移植进行了未充分缝合的结直肠吻合。这项新的应用表明, ASC 片可以减少结直肠吻合口漏。

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

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

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

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

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 ...

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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 ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

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

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

部分原位肝移植大鼠模型的肝动脉重建的外科手术

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

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

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

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

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

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

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

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

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 ...

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

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

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

静脉介入型号:研究旁路移植通畅一个合适的模型

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

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

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

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

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 ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

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

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

肝细胞部分肝切除术后脾移植。小鼠

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

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

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

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

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

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

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