作者感谢再生医学的维克森林大学的帮助与此协议的开发。我们从大奥蒙德街医院慈善助学金承认的支持，该基金会欧亨尼奥Litta的（瑞士日内瓦），医学研究理事会，英国皇家外科医学院英格兰，火花儿童医疗慈善机构，英国外交部对英国/美国干细胞协同合作奖和米塔尔研究基金。我们还要感谢英国皇家学会/沃尔夫森基金会在儿童健康研究所获得的小儿外科组织工程实验室整修补助金。 PDC和东南由大奥蒙德街医院儿童慈善活动的支持。

1。大鼠肠隔离<ol><li>制备磷酸盐缓冲盐水（Sigma公司，英国）的1升溶液中含有1％抗生素/抗真菌剂（Sigma公司，英国）（PBS / AA）。适合蠕动泵（的Masterflex L / S变速）与两个管子。通过蠕动泵15分钟，在最大速度下的管中，然后PBS / AA另外15分钟运行70％乙醇（EtOH中）。 </li><li>安乐死成年SD大鼠的CO 2吸入并根据当地动物的指引及批准（在英国，在动物（科学规程）行动1986年内政部指引）颈椎脱臼重约250-350克。 </li><li>使用前高压灭菌的手术器械。喷雾和擦拭用70％乙醇的动物的腹部。 </li><li>执行上腹部xifo-耻骨开腹切口，以保证术野清晰。 </li><li>剔骨右侧动物的小肠到纸巾上喷以70％的乙醇。德ë护理连续润湿，用PBS / AA肠腔从而避免细胞外基质（ECM）的组织和变性的脱水。 </li><li>定位肠系膜上动脉（SMA）在从朝向肠主动脉成90°角而产生。使用一个27G的套管从主动脉进入SMA和迅速退出针以避免撕裂容器的壁。推进套管的塑料管插入SMA和安全使用缝线（薇乔5/0）。 </li><li>通过注入1ml的PBS / AA的确认插管。使用弹簧剪刀切开主动脉近端和远端向SMA以便释放插管。 </li><li>避免粪便泄漏解剖出大肠时，放置在回盲结的近端1（丝5/0）缝合线结，一个在乙状结肠的远端。然后切两个结之间的回肠末端和结肠并取出大肠。 </li><li>切开肠管在空肠 - 十二指肠交界处ND释放肠任何结缔组织连接，它可能与腹部。 </li><li>转让在培养皿用PBS / AA小肠。导管插入肠的近端部分有一个塑料巴斯德吸管（LSL实验室消耗品），并用缝合线将其固定到位。切吸液管，因此将适合露儿乐60 ml注射器中（BD Plastipak）。冲洗肠腔用60毫升的PBS / AA的2倍，除去粪便和杂物。 </li></ol> 2。脱细胞方法<ol><li>血管插管连接到三通旋塞阀。这将缓解处理，尽量减少紧张局势血管树，并允许去除气泡从管子。 </li><li>开始通过的Masterflex L / S变速滚轮泵为0.6毫升/小时运行去离子水（电阻率18.2MΩ/平方厘米）。确保无气泡与肠腔旋塞阀连接之前，内管存在。一些泡沫可能是从步骤管腔内本1.9，这可能会损害脱细胞过程。改变泵的速度和处理组织钳小心，以确保气泡出口的远端。直到这个过程结束，因为高转速可能会损坏血管树不要连接血管插管。 </li><li>传送装置向冷室中，由于在4℃下进行脱细胞的接下来的步骤在较低温度时允许去除细胞的同时最大限度地减少组织分解。将培养皿在一个容器，将收集四溢的解决方案。 24小时为0.6毫升/小时灌注两肠腔和血管树，用去离子水（电阻率18.2MΩ/平方厘米）。对于第一个小时定期检查设备，以确保所有的连接牢固，漏电极小，无气泡是肠道内出现。 </li><li>后，用去离子水治疗24小时，脱细胞移动设备中的冷室作为步骤外其余在室温（RT）下进行。 </li><li>权衡脱氧胆酸钠（Sigma公司，英国）以制备300毫升在去离子水中的4％溶液。称取去氧胆酸钠吸气罩下，因为它是一种口服和眼睛有刺激性。混合使用涡旋直至溶液澄清。可将溶液保持在4℃下长达1个月。 </li><li>改变去离子解决脱氧胆酸钠，请检查连接，消除任何气泡和灌注0.6毫升/小时4小时。 </li><li>以下的脱氧胆酸钠步骤洗涤，用PBS / AA 30分钟，以避免脱氧胆酸钠和DNA酶-I之间的相互作用。 </li><li>称出脱氧核糖核酸酶-I（DNA酶I，Sigma）和准备在1M氯化钠，pH值7.4加入250毫升一2000 KU溶液。混合使用涡旋直至溶液澄清。该溶液必须在使用前立即制备，并可以不被存储。 </li><li>灌注用DNase-I为3小时，在室温下以0.6毫升/小时的速度。 </li><li>随着DNA酶-I步，转移脚手架成组织培养罩和改变板和仪器。它建议使用无菌技术。 </li><li>用PBS / AA 30分钟洗，以保证脱细胞解决方案的所有残余都被删除。将支架在一个新的培养皿中，并转移到一个UV交联剂（SPECTROLINE，美国）。运行两个灭菌循环。存放在5％的PBS / AA和更改解​​决方案每3天。 </li></ol>

<ol>
	<li>Atala, A., Bauer, S. B., Soker, S., Yoo, J. J., Retik, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+autologous+bladders+for+patients+needing+cystoplasty.">Tissue-engineered autologous bladders for patients needing cystoplasty.</a> Lancet. 367 (9518), 1241-1246 (2006).</li><li>Raya-Rivera, A., Esquiliano, D. R., Yoo, J. J., Lopez-Bayghen, E., Soker, S., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+autologous+urethras+for+patients+who+need+reconstruction:+an+observational+study.">Tissue-engineered autologous urethras for patients who need reconstruction: an observational study.</a> Lancet. 377 (9772), 1175-1182 (2011).</li><li>Macchiarini, P., Jungebluth, 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=Clinical+transplantation+of+a+tissue-engineered+airway.">Clinical transplantation of a tissue-engineered airway.</a> Lancet. 372 (9655), 2023-2030 (2008).</li><li>Macchiarini, P., Walles, T., Biancosino, C., Mertsching, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+human+transplantation+of+a+bioengineered+airway+tissue.">First human transplantation of a bioengineered airway tissue.</a> J. Thorac. Cardiov. Surg. 128 (4), 638-641 (2004).</li><li>Elliott, M. J., de Coppi, 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=Stem-cell-based,+tissue+engineered+tracheal+replacement+in+a+child:+a+2-year+follow-up+study.">Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study.</a> Lancet. 380 (9846), 994-1000 (2012).</li><li>Grikscheit, T. C., Siddique, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection.">Tissue-engineered small intestine improves recovery after massive small bowel resection.</a> Ann. Surg. 240 (5), 748-754 (2004).</li><li>Sala, F. G., Matthews, J. A., Speer, A. L., Torashima, Y., Barthel, E. R., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multicellular+Approach+Forms+a+Significant+Amount+of+Tissue-Engineered+Small+Intestine+in+the+Mouse.">A Multicellular Approach Forms a Significant Amount of Tissue-Engineered Small Intestine in the Mouse.</a> Tissue Eng. Part A. , (2011).</li><li>Matthews, J. A., Sala, F. G., Speer, A. L., Warburton, D., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+optimizes+the+formation+of+tissue-engineered+small+intestine.">VEGF optimizes the formation of tissue-engineered small intestine.</a> Regen. Med. 6 (5), 559-567 (2011).</li><li>Chen, M. K., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+bowel+tissue+engineering+using+small+intestinal+submucosa+as+a+scaffold.">Small bowel tissue engineering using small intestinal submucosa as a scaffold.</a> J. Surg. Res. 99 (2), 352-358 (2001).</li><li>Wang, Z. Q., Watanabe, Y., Toki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+assessment+of+small+intestinal+submucosa+as+a+small+bowel+graft+in+a+rat+model.">Experimental assessment of small intestinal submucosa as a small bowel graft in a rat model.</a> J. Pediatr. Surg. 38 (11), 1596-1601 (2003).</li><li>Wang, Z. Q., Watanabe, Y., Noda, T., Yoshida, A., Oyama, T., Toki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+evaluation+of+regenerated+small+bowel+by+small+intestinal+submucosa.">Morphologic evaluation of regenerated small bowel by small intestinal submucosa.</a> J. Pediatr. Surg. 40 (12), 1898-1902 (2005).</li><li>Hori, Y., Nakamura, 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=Experimental+study+on+tissue+engineering+of+the+small+intestine+by+mesenchymal+stem+cell+seeding.">Experimental study on tissue engineering of the small intestine by mesenchymal stem cell seeding.</a> J. Surg. Res. 102 (2), 156-160 (2002).</li><li>Gardner-Thorpe, J., Grikscheit, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+tissue-engineered+small+intestine.">Angiogenesis in tissue-engineered small intestine.</a> Tissue Eng. 9 (6), 1255-1261 (2003).</li><li>Conconi, M. T., Coppi, P. D., 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=Tracheal+matrices,+obtained+by+a+detergent-enzymatic+method,+support+in+vitro+the+adhesion+of+chondrocytes+and+tracheal+epithelial+cells.">Tracheal matrices, obtained by a detergent-enzymatic method, support in vitro the adhesion of chondrocytes and tracheal epithelial cells.</a> Transpl. Int. 18 (6), 727-734 (2005).</li><li>Ott, H. C., Matthiesen, T. 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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. 14 (2), 213-221 (2008).</li><li>Uygun, B. E., Soto-Gutierrez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat. Med. , 1-8 (2010).</li><li>Uygun, B. E., Price, 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=Decellularization+and+recellularization+of+whole+livers.">Decellularization and recellularization of whole livers.</a> J. Vis. Exp. (48), e2394 (2011).</li><li>Petersen, T. H., Calle, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Engineered+Lungs+for+in+Vivo+Implantation.">Tissue-Engineered Lungs for in Vivo Implantation.</a> Science. 329 (5991), 538-541 (2010).</li><li>Ott, H. C., Clippinger, B., 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=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16 (8), 927-933 (2010).</li><li>Calle, E. A., Petersen, T. H., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+lung+engineering.">Procedure for lung engineering.</a> J. Vis. Exp. (49), e2651 (2011).</li><li>Totonelli, G., Maghsoudlou, 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=A+rat+decellularized+small+bowel+scaffold+that+preserves+villus-crypt+architecture+for+intestinal+regeneration.">A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration.</a> Biomaterials. 33 (12), 3401-3410 (2012).</li><li>Sala, F. G., Kunisaki, S. M., Ochoa, E. R., Vacanti, J., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+small+intestine+and+stomach+form+from+autologous+tissue+in+a+preclinical+large+animal+model.">Tissue-engineered small intestine and stomach form from autologous tissue in a preclinical large animal model.</a> J. Surg. Res. 156 (2), 205-212 (2009).</li><li>Demirbilek, S., Kanmaz, T., Ozardali, I., Edali, M. N., Y&#252;cesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+porcine+small+intestinal+submucosa+in+intestinal+regeneration.">Using porcine small intestinal submucosa in intestinal regeneration.</a> Pediatr. Surg. Int. 19 (8), 588-592 (2003).</li></ol>

没有利益冲突的声明。

设立这个实验最困难的步骤涉及SMA的插管，并建立和维持无菌的。 Cannulating在SMA在啮齿类动物中而不破裂的壁可以是由于容器的尺寸和位置相当困难的。可选地，缝合线可被置于围绕所述近端主动脉前向SMA原点，其次是主动脉本身远侧插管，指挥塑料插管插入SMA。在脱细胞可怜的导管放置和高流速的组合可能导致丢失的血管通路。不育是由于存在于小肠菌群的数量的一个重大问题。以下收获用PBS洗涤/ AA是非常重要的，并且排泄物或碎屑的任何迹象，必须从腔中移除。在falcon管中，用DMEM将所述支架的一部分在孵化以下紫外线消毒应是一个指标，如果不育已经实现。在该情况下，细菌定植，媒体将改变pH值与来自红的颜色转向黄色。为了解决这个问题，进一步紫外线周期，建议，以及用PBS洗涤含有高浓度的抗生素/抗真菌的。 一个可能的修改以获得既血管和静脉通路支架是将导管插入下腔静脉（IVC），以及对SMA。从静脉血管两侧脱细胞应间歇地尝试所有三种解决方案。连续脱细胞可以由具有在两侧的正压​​力脉冲串的毛细血管。 多年来已经出现了许多体外和体内 6,9,12使用不同的细胞支架材料的组合在肠TE努力。大部分工作已经使用涂有胶原蛋白I型6,7,13,22管状非织造布95％的PGA-5％PLGA支架进行。下面播种肠上皮组织体单位（OU），并在一段植入到小鼠的网膜时，它们形成囊肿肌肉上，然后可以卷管内侧到外侧和上皮。然而，在这些支架的设计的孔隙率和简洁不允许用于在体外环境中，例如，一个生物反应器中的大颗人造肠的产生。此外，由于缺乏可连接到接收方的进一步的先天血管网的限制使用这种支架的临床平移。除了与PGA-PLGA支架的实验中，其它基团所用的胶原蛋白或SIS的支架，这两者不复制肠道的复杂性。 SIS尤其是肠组织工程的唯一一次公布的方法，并已应用于150多个医疗环境，展示脱细胞支架，以提供机械稳定性和促进细胞生长，而铅的能力ing到无免疫原性反应。然而，由于缺乏适当的宏观和微架构，导致了其"用于肠道TE的目的9-11,23效果差。 的脱细胞的方法，我们已经描述的优点包括微观特征，如管腔隐窝 - 绒毛体系结构表示为复育适当的环境中被肠干细胞小生境的保存。在宏观方面，一种分级的血管网的存在下，使附着到接收者，从而提供营养和氧气到TE-肠管21的所有层。更重要的是，细胞外基质组分如胶原，弹性蛋白和glyocosaminoglycans的维护不仅对机械特性，而且还指示细胞增殖和分化中起重要作用。最重要的是，脱细胞的方法的能力被放大到大Ř组织，同时保持相同的特性是用于TE的临床翻译的重要特征。 天然肠基质用血管网络的发展允许创建较大的人造肠，可连接到主机的段。

组织工程（TE）提供了一个治疗选择器官移植，绕过免疫抑制和器官短缺的问题。 TE最近有在临床的成功应用，具有替代器官，如膀胱1，尿道口2和气管，无论是在3,4成人和儿童5。 构建组织工程化器官需要一个支架与适当的细胞相结合。甲支架可以使用天然来源的（ 如胶原蛋白）进行制备和合成的（ 例如，聚-L-羟基乙酸; PLGA）的材料，或由天然器官和组织的脱细胞来获得。已经使用的迄今为肠TE支架主要被任一脱细胞（小肠粘膜下层）或合成的（聚-L-羟基乙酸和聚乳酸）6-13。这些生物材料是很简单的在宏观和微观架构，可能不是很理想，如果组织工程小肠被临床换算。对于肠道的最佳生物材料应具有一种天生的血管树，可连接到主机的血液供应，分层管状壁具有不同的属性，以反映对肠壁的各层和绒毛隐窝轴线上的腔侧，以帮助用再增殖上皮干细胞。 脱细胞是从整个器官取出细胞，同时保持其原有的建筑14棚架产生了一种新的方法。这是优选的，以现有的支架，因为它们不仅复制该器官的结构，而且包含嵌入的细胞外基质（ECM），该辅助细胞的增殖和分化中的化学信号。在2008年一个尸体气管脱细胞14，播种与患者自身的细胞，并移植到更换主力左支气管的年轻人<sup&gt; 3。从那时起，一些团体已经报道的生产脱细胞支架在小型和大型动物的心脏15，16,17肝和肺18-20。 我们还适于相同的方法，以产生一个小肠粘膜脱细胞的支架21。本文所描述的方法的目的是产生一个保持原有的组织，例如血液供应，以及绒毛隐窝轴在肠腔内的微观结构的宏观特征脱细胞的肠矩阵。我们相信，这种方法可能最终会采用其他器官，以提高脱细胞的效率。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ethanol solution, 70% in H20</td>
			<td>Sigma</td>
			<td>02877</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline tablets</td>
			<td>Sigma</td>
			<td>79382</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100x)</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td>Oral and eye irritant; use protection</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
	</tbody>
</table>

a decellularization methodology for the production of a natural acellular intestinal matrix

成功的组织工程支架包括与适当的细胞在体外或在体内的结合。支架可以是合成的，天然衍生的或从组织/器官中得到。后者使用的是所谓的脱细胞技术来获得。脱细胞可能涉及的物理，化学和酶相结合的方法。该技术的目标是消除所有蜂窝痕迹，同时保持原始组织的宏观和微观结构。 肠组织工程迄今已使用相对简单的支架不复制原生器官的复杂结构。本文的重点是描述对大鼠小肠一种有效的脱细胞技术。小肠的隔离，以保证血管连接的维护进行说明。化学和酶溶液的组合，以除去细胞whilST保留绒毛隐窝轴支架的管腔方面亦载。最后，制作支架的相应特性的评估进行了讨论。

在成功脱细胞（ 图1），该支架应该具有宏观外观类似于天然肠中的一个，但具有半透明的壁（ 图2A）。宏观架构的保存也应该由血管树的通畅性是显而易见的。当染料是通过SMA插管注射，应该均匀地分布在整个支架和到达毛细管树（ 图2B）。棚架的结构应该是免费的细胞核和细胞质材料，东西，可以使用DNA定量试剂盒和简单的组织学分析进行评估。苏木精伊红染色（H＆E）应表现出缺乏的细胞核和细胞质物质，同时保留小肠层（ 图3A）。特别是，在腔侧的绒毛和隐窝中的维护应明显下列扫描电子显微镜（ 图4）。一保护细胞外基质组分如胶原，弹性蛋白和粘多糖的还应预期。例如，在所述支架（ 图3B）马松三色染色显示完整的结缔组织。 <img alt="图1" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig1.jpg" width="600px" /> 图1。实验计划时间表的脱细胞处理程序; PBS / AA:磷酸盐缓冲液与抗生素抗真菌，NaDeoxy:脱氧胆酸钠，RT:室温，DNA酶-I:脱氧核糖核酸酶-I <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50658/50658fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig2.jpg" width="600px" /> 图2。脚手架macroscopiC特征。大鼠小肠下成功脱细胞肉眼外观（A）。注射罗索丽春红染过SMA的演示了一个完整的血管网（B）平均:肠系膜上动脉<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50658/50658fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig3.jpg" width="600px" /> 图3。 。组织学H＆E（A）和MT（B）染色表明缺乏与保存结缔组织结构的细胞物质，H＆E:苏木精伊红，MT:Masson染色。比例尺:100微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50658/50658fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 </P&gt; <img alt="图4" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig4.jpg" width="600px" /> 图4。 。电子显微镜扫描电子显微镜表示保留绒毛，隐窝结构的支架内腔;标尺:100微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50658/50658fig4highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix at JoVE.com

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

本文介绍了用于生产从大鼠小肠脱细胞基质的方法。肠道支架的推导是重要的组织工程未来的应用，干细胞生物学和药物测试。

一脱细胞方法对天然脱细胞基质肠的生产

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

a-decellularization-methodology-for-production-natural-acellular

Nanyang Technological University

Research

JoVE Journal

Bioengineering

29.1K 次观看.  University College London. 本文介绍了用于生产从大鼠小肠脱细胞基质的方法。肠道支架的推导是重要的组织工程未来的应用，干细胞生物学和药物测试。 

视频: A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

整个肝脏的脱细胞和Recellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

科研

生物工程

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

人隐静脉的去细胞和 Recellularization 方法学研究

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

倒置方向下整个人的心脏在加压袋内的去细胞化

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

全猪胰腺的分离与去细胞

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...