Name: decellularization of whole human heart inside a pressurized pouch in an inverted orientation
Uploaded: 2018-11-26T13:30:00
Description: Watch this Scientific Journal Video about Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation at JoVE.com

这项研究得到了休斯敦捐赠赠款和德州新兴技术基金的支持。作者感谢器官采购机构 life赠送, inc. 和捐献者的家人使这项研究成为可能。

所有实验都遵循了德克萨斯心脏研究所的道德委员会准则。
1. 器官准备
注: 与德克萨斯州 (http://www.lifegift.org) 的非营利器官采购组织 life赠送组织合作, 在获得批准的情况下, 捐赠了不适合移植的人的心脏进行研究。
<ol><li>为了获得心脏, 静脉注射 30, 000 u 肝素到心脏。安全地缝合主动脉中的心绞痛插管, 并附加一条夹紧灌注线。穿孔下腔静脉 (ivc) 排出右?...

<ol>
	<li>Writing Group Members. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+Summary:+Heart+Disease+and+Stroke+Statistics--2016+Update:+A+Report+From+the+American+Heart+Association.">Executive Summary: Heart Disease and Stroke Statistics--2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), 447-454 (2016).</li><li>Zia, 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=Hearts+beating+through+decellularized+scaffolds:+whole-organ+engineering+for+cardiac+regeneration+and+transplantation.">Hearts beating through decellularized scaffolds: whole-organ engineering for cardiac regeneration and transplantation.</a> Critical Reviews in Biotechnology. 36 (4), 705-715 (2016).</li><li>Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strip+and+Dress+the+Human+Heart.">Strip and Dress the Human Heart.</a> Circulation Research. 118 (1), 12-13 (2016).</li><li>Ott, H. 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=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> Nature Medicine. 14 (2), 213-221 (2008).</li><li>Sanchez, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+human+heart+matrix:+A+critical+step+toward+whole+heart+grafts.">Acellular human heart matrix: A critical step toward whole heart grafts.</a> Biomaterials. 61, 279-289 (2015).</li><li>Peloso, 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=Current+achievements+and+future+perspectives+in+whole-organ+bioengineering.">Current achievements and future perspectives in whole-organ bioengineering.</a> Stem Cell Research &amp; Therapy. 6, 107 (2015).</li><li>Guyette, J. 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=Perfusion+decellularization+of+whole+organs.">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</li><li>Momtahan, N., Sukavaneshvar, S., Roeder, B. L., Cook, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+Processes+to+Decellularize+and+Cellularize+Hearts+to+Generate+Functional+Organs+and+Reduce+the+Risk+of+Thrombosis.">Strategies and Processes to Decellularize and Cellularize Hearts to Generate Functional Organs and Reduce the Risk of Thrombosis.</a> Tissue Engineering Part B-Reviews. 21 (1), 115-132 (2015).</li><li>Lee, P. 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=Inverted+orientation+improves+decellularization+of+whole+porcine+hearts.">Inverted orientation improves decellularization of whole porcine hearts.</a> Acta Biomaterialia. , (2016).</li><li>Momtahan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automation+of+Pressure+Control+Improves+Whole+Porcine+Heart+Decellularization.">Automation of Pressure Control Improves Whole Porcine Heart Decellularization.</a> Tissue Eng Part C Methods. , (2015).</li><li>Weymann, 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=Development+and+Evaluation+of+a+Perfusion+Decellularization+Porcine+Heart+Model+-+Generation+of+3-Dimensional+Myocardial+Neoscaffolds.">Development and Evaluation of a Perfusion Decellularization Porcine Heart Model - Generation of 3-Dimensional Myocardial Neoscaffolds.</a> Circulation Journal. 75 (4), 852-860 (2011).</li><li>Weymann, 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=Bioartificial+heart:+A+human-sized+porcine+model--the+way+ahead.">Bioartificial heart: A human-sized porcine model--the way ahead.</a> PLoS One. 9 (11), e111591 (2014).</li><li>Remlinger, N. T., Wearden, P. D., Gilbert, T. W. <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+decellularization+of+porcine+heart+by+retrograde+coronary+perfusion.">Procedure for decellularization of porcine heart by retrograde coronary perfusion.</a> Journal of Visualized Experiments. (70), e50059 (2012).</li><li>Guyette, J. 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=Bioengineering+Human+Myocardium+on+Native+Extracellular+Matrix.">Bioengineering Human Myocardium on Native Extracellular Matrix.</a> Circulation Research. 118 (1), 56-72 (2016).</li><li>Sanchez, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+from+acellular+human+heart+matrix.">Data from acellular human heart matrix.</a> Data Brief. 8, 211-219 (2016).</li><li>Garreta, E., 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=Myocardial+commitment+from+human+pluripotent+stem+cells:+Rapid+production+of+human+heart+grafts.">Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts.</a> Biomaterials. 98, 64-78 (2016).</li><li>Wainwright, 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=Preparation+of+Cardiac+Extracellular+Matrix+from+an+Intact+Porcine+Heart.">Preparation of Cardiac Extracellular Matrix from an Intact Porcine Heart.</a> Tissue Engineering Part C-Methods. 16 (3), 525-532 (2010).</li><li>Larson, A. M., Yeh, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+characterization+of+sub-10-fs+pulses.">Ex vivo characterization of sub-10-fs pulses.</a> Optics Letters. 31 (11), 1681-1683 (2006).</li><li>Lee, P. F., Yeh, A. T., Bayless, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+microscopy+reveals+invading+endothelial+cells+anisotropically+alter+three-dimensional+collagen+matrices.">Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices.</a> Experimental Cell Research. 315 (3), 396-410 (2009).</li><li>Lee, P. F., Bai, Y., Smith, R. L., Bayless, K. J., Yeh, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+responses+are+enhanced+in+mechanically+and+microscopically+characterized,+microbial+transglutaminase+crosslinked+collagen+matrices+with+increased+stiffness.">Angiogenic responses are enhanced in mechanically and microscopically characterized, microbial transglutaminase crosslinked collagen matrices with increased stiffness.</a> Acta Biomaterialia. 9 (7), 7178-7190 (2013).</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=Multi-photon+microscopy+in+cardiovascular+research.">Multi-photon microscopy in cardiovascular research.</a> Methods. 130, 79-89 (2017).</li><li>Ramanathan, T., Skinner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+blood+flow.">Coronary blood flow.</a> Continuing Education in Anaesthesia Critical Care &amp; Pain. 5 (2), 61-64 (2005).</li><li>Murthy, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Quantification+of+Myocardial+Blood+Flow+Using+PET:+Joint+Position+Paper+of+the+SNMMI+Cardiovascular+Council+and+the+ASNC.">Clinical Quantification of Myocardial Blood Flow Using PET: Joint Position Paper of the SNMMI Cardiovascular Council and the ASNC.</a> Journal of Nuclear Cardiology. 25 (1), 269-297 (2018).</li><li>Molina, D. K., DiMaio, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+organ+weights+in+men:+Part+I-the+heart.">Normal organ weights in men: Part I-the heart.</a> The American Journal of Forensic Medicine and Pathology. 33 (4), 362-367 (2012).</li><li>Molina, D. K., DiMaio, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+Organ+Weights+in+Women:+Part+I-The+Heart.">Normal Organ Weights in Women: Part I-The Heart.</a> The American Journal of Forensic Medicine and Pathology. 36 (3), 176-181 (2015).</li><li>Robertson, M. J., Dries-Devlin, J. L., Kren, S. M., Burchfield, J. S., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+cellularization+of+whole+decellularized+heart+extracellular+matrix.">Optimizing cellularization of whole decellularized heart extracellular matrix.</a> PLoS One. 9 (2), e90406 (2014).</li><li>Robertson, M. J., Soibam, B., O'Leary, J. G., Sampaio, L. C., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellularization+of+rat+liver:+An+in+vitro+model+for+assessing+human+drug+metabolism+and+liver+biology.">Cellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.</a> PLoS One. 13 (1), e0191892 (2018).</li><li>Baghalishahi, 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=Cardiac+extracellular+matrix+hydrogel+together+with+or+without+inducer+cocktail+improves+human+adipose+tissue-derived+stem+cells+differentiation+into+cardiomyocyte-like+cells.">Cardiac extracellular matrix hydrogel together with or without inducer cocktail improves human adipose tissue-derived stem cells differentiation into cardiomyocyte-like cells.</a> Biochemical and Biophysical Research Communications. , (2018).</li><li>Perea-Gil, 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=In+vitro+comparative+study+of+two+decellularization+protocols+in+search+of+an+optimal+myocardial+scaffold+for+recellularization.">In vitro comparative study of two decellularization protocols in search of an optimal myocardial scaffold for recellularization.</a> American Journal of Translational Research. 7 (3), 558-573 (2015).</li><li>Freytes, D. O., O'Neill, J. D., Duan-Arnold, Y., Wrona, E. A., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+cardiac+extracellular+matrix+hydrogels+for+cultivation+of+human+stem+cell-derived+cardiomyocytes.">Natural cardiac extracellular matrix hydrogels for cultivation of human stem cell-derived cardiomyocytes.</a> Methods Molecular Biology. 1181, 69-81 (2014).</li><li>Oberwallner, 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=Preparation+of+cardiac+extracellular+matrix+scaffolds+by+decellularization+of+human+myocardium.">Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium.</a> Journal of Biomedical Materials Research Part A. 102 (9), 3263-3272 (2014).</li></ol>

据我们所知, 这是首次报告在加压袋内的人的心脏倒置去病毒化, 并对流速和细胞碎片清除进行延时监测。心包状袋在整个去渗过程中保持心脏方向稳定。与传统的直立朗根多夫灌注解离方法9相比, 将整个心脏浸入眼袋内可防止脱水, 并最大限度地减少主动脉 (从心脏重量) 上的过度拉伤.这反过来又保持了主动脉瓣的能力。
为了实现最大的去渗效率, 冠状动脉?...

心力衰竭导致患者死亡率高。终末期心力衰竭的最终治疗方案是同种异体移植。然而, 由于供体器官短缺, 移植的等待列表很长, 患者面临着从终身免疫抑制到慢性器官排斥1,2的移植后障碍。生物工程功能性心脏通过用病人自己的细胞重新填充去细胞化的人类大小的心脏, 可以绕过这些障碍3。
"工程" 心脏的一个重要步骤是创建一个脚手架, 具有适当的血管和实质结构、组成和功能, 以指导所交付细胞的排列和组织。在适当的框架存在的情况下, 在脚手架上播种的细胞应能识别环境, 并作为该器官的一部分发挥预期的功能。在我们看来, 去细胞外基质 (decm) 包括理想支架的必要特性。
通过利用内在血管, 可以通过前置或逆行灌注4来去除细胞成分, 同时保留微妙的三维细胞外基质和血管 2, 5,6,7。功能血管在生物工程全器官中就像在体内一样, 对营养分配和废物清除都很重要。冠状动脉灌注去细胞减少已被证明是有效的创造去细胞化的心脏从大鼠4,或猪4,7, 9, 10,11 12, 13,和人类5,7, 14,15,16。然而, 阀门、心房和其他 "薄" 区域的完整性可能会受到影响。
用压力控制7、9、10、11、12或输液流量控制13 ,17和来自人类捐献者使用压力控制5,7, 14,15。人类供体心脏去细胞化发生在4-8 内, 压力控制在 0-100毫米汞柱,垂直方向5、15、16或16天以上, 压力控制在60毫米汞柱14.在压力控制的前去室化条件下, 主动脉瓣的能力对保持主动脉根部冠状动脉灌注效率和稳定压力起着至关重要的作用。我们以前的工作表明, 心脏的方向影响其冠状动脉灌注效率在脱肺过程中, 因此在最后9的脚手架完整性.
作为我们以前工作的延续 9, 我们引入了一个新的概念, 其中一个心包样袋添加, 以提高全心去渗。我们描述了人类心脏被放置在加压袋内的去细胞化, 反向定向, 主动脉根部的压力控制在120毫米汞柱。该协议包括监测整个去室化过程中的流动剖面和流出介质的收集, 以评估冠状动脉灌注效率和细胞碎片清除。然后进行生化检测, 以测试该方法的有效性。

<table>
	<tbody>
		<tr>
			<td>2-0 silk suture</td>
			<td>Ethicon</td>
			<td>SA85H</td>
			<td>Suture used to ligate superior and inferior vena cava</td>
		</tr>
		<tr>
			<td>1/4&#34; x 3/8&#34; connector with luer</td>
			<td>NovoSci</td>
			<td>332023-000</td>
			<td>Connect aorta and pulmonary artery</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing</td>
			<td>Cole-Parmer</td>
			<td>HV-96410-16</td>
			<td>Tubing to connect heart chambers/veins</td>
		</tr>
		<tr>
			<td>infusion and outflow line</td>
			<td>Smiths Medical</td>
			<td>MX452FL</td>
			<td>For flowing solutions through the vasculature</td>
		</tr>
		<tr>
			<td>Polyester pouch (Ampak 400 Series SealPAK Pouches)</td>
			<td>Fisher scientific</td>
			<td>01-812-17</td>
			<td>Pericardium-like pouch for containing heart during decellularization</td>
		</tr>
		<tr>
			<td>Snapware Square-Grip Canister</td>
			<td>Snapware</td>
			<td>1022</td>
			<td>1-liter Container used for perfusing heart</td>
		</tr>
		<tr>
			<td>Black rubber stoppers</td>
			<td>VWR</td>
			<td>59586-162</td>
			<td>To seal the perfusion container</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Harvard Apparatus</td>
			<td>881003</td>
			<td>To pump fluid through the inflow lines and to drain fluids</td>
		</tr>
		<tr>
			<td>2 L aspirator bottle with bottom sidearm</td>
			<td>VWR</td>
			<td>89001-532</td>
			<td>For holding solutions/perfusate</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay kit</td>
			<td>Life Technologies</td>
			<td>P7589</td>
			<td>For quantifying dsDNA</td>
		</tr>
		<tr>
			<td>Calf thymus standard</td>
			<td>Sigma</td>
			<td>D4522</td>
			<td>DNA standard</td>
		</tr>
		<tr>
			<td>Blyscan Glycosaminoglycan Assay Kit</td>
			<td>Biocolor Ltd</td>
			<td>Blyscan #B1000</td>
			<td>GAG assay kit</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Tecan</td>
			<td>Infinite M200 Pro</td>
			<td>For analytical assays</td>
		</tr>
		<tr>
			<td>GE fluoroscopy</td>
			<td>General Electric</td>
			<td>OEC 9900 Elite</td>
			<td>Angiogram</td>
		</tr>
		<tr>
			<td>Visipaque</td>
			<td>GE</td>
			<td>13233575</td>
			<td>Contrast agent</td>
		</tr>
	</tbody>
</table>

decellularization of whole human heart inside a pressurized pouch in an inverted orientation

终末期心力衰竭患者的最终解决方案是器官移植。但供体心脏是有限的, 免疫抑制是必需的, 并且拒绝可能发生。创建一个功能性的、自体的生物人工心脏可以解决这些挑战。由脚手架和细胞组成的心脏的生物制造是一种选择。一个天然的支架与组织特异性组成以及微观和宏观架构可以获得通过去细胞化的心脏从人类或大型动物, 如猪。去光化包括在保持3d 细胞外基质和血管的同时冲洗细胞碎片, 并允许在以后的时间点进行 "纤维素化"。利用我们的新发现, 复杂器官的灌注解离是可能的, 我们开发了一种更 "生理" 的方法, 通过将非可移植的人的心脏放置在一个加压袋, 在一个倒置的方向, 在可控压力下。使用加压袋的目的是在主动脉瓣上产生压力梯度, 以保持其关闭状态, 并改善心肌灌注。在去室去光化过程中同时评估流动动力学和细胞碎片清除, 使我们能够监测流体流入和碎片流出, 从而产生一个脚手架, 可用于简单的心脏修复 (例如, 作为一个补丁或阀支架) 或作为全器官脚手架。

在120毫米汞柱的恒压下, 经过7天的主动脉前灌注去细胞化, 人的心脏变成了半透明 (图 6b)。心脏被严重解剖成19个部分进行生化 (dna、gag 和 sds) 分析 (图 6c), 以评估最终脱病毒产物。
在整个去电磁化过程中, 不同溶液的输液流量随压力保持不变而变化。当灌注溶液从高渗变为低渗溶液...

Watch this Scientific Journal Video about Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation at JoVE.com

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

这种方法可以使用基于渗透冲击的简单协议和最小器官破坏矩阵的离子洗涤剂灌注, 使复杂的固体器官去极化。它包括一种新的人的心脏在加压袋内的去电磁化技术, 并实时监测流动动力学和细胞碎片流出。

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 ...

这种方法可以帮助回答有关产生血管化脚手架的关键问题，而血管化脚手架是组织工程领域的圣杯。这项技术的主要优点是，倒置方向的加压袋可改善不可移植人体器官的去细胞化，并使我们能够在适当的时间范围内以无菌的方式做到这一点。要让心脏做好去细胞化的准备，请首先对可能的缺陷进行内部检查。如果存在隔膜缺陷，请用适当的缝合线纠正缺陷。接下来，用2-0的丝绸缝合来将上等的维纳卡瓦斯拉下马。用 5-0 PROLENE 缝合右中庭壁，并解剖远离主肺动脉的主动脉，以便随后进行管状。根据容器的直径将连接器插入主动脉和肺动脉，用 2-0 丝线固定连接器。使用其中一个肺静脉孔，通过左中庭向左心室插入一条输油管管线，并将输液管线连接到主动脉中的连接器，将流出线连接到肺动脉中的连接器。将准备好的心脏放入倒置方向的聚酯袋中，然后将袋放入灌注容器中。根据容器的直径，将每条管路连接到橡胶塞子中各端口，并将塞子插入灌注容器的盖子中，以密封聚酯袋。然后，通过橡胶塞的输液口对PBS进行灌注，以验证从肺动脉和插入左心室的管路流出，利用水流清除血管内任何残留的血液痕迹。心脏准备就绪后，将组装的生物反应器垂直放置，将输液管路、压力头线、肺动脉流出线和生物反应器排空管线连接到灌注生物反应器顶部的橡胶盖表面端口。然后，用超音速溶液去细胞心脏四小时，用低度溶液解2小时，用硫酸钠（SDS）120小时，最后用120升PBS洗涤，所有洗涤量都在音管根部测得的恒定120毫米汞压下。在PBS洗涤的最后10升中，加入500毫升无菌2.1%的过乙酸溶液，用10种正常氢氧化钠中和到灌注溶液中，对脚手架进行消毒。通常，在去细胞化过程中进入大母的流速会随着灌注溶液从超音速到低吨位的变化而逐渐减少。相比之下，当灌注溶液从低度溶液更改为 SDS 时，流速增加，此时输液流速表现出波动。肺动脉的流出率与高、低氧溶液呈类似趋势。然而，SDS灌注期间的流出率总体下降。冠状动脉灌注效率也随着时间的推移而降低，因为不同的试剂通过血管注入。由于肺动脉和左心室的外流呼吸器同时收集，可以通过光谱分析比较其碎片含量。在灌注期间，两个血管的污水的浊度会随着时间的推移而减小，尽管肺动脉的浊度与最初灌注期间从左心室观察到的颜色变化相比，呈现出更突然的颜色变化。通过六颗脱细胞人类心脏的双子酸蛋白测定，对流出浊度与细胞碎片之间的相关性进行评估，揭示了蛋白质浓度与流出浊度之间的线性相关性。在尝试此过程时，必须监控流速并定期收集灌注，以实际监控灌注过程。按照这个程序，其他技术，如机械评估或无菌测试可用于评估脚手架的保真度及其潜在的用途，重新细胞生成功能性心脏组织。该技术开发后，为再生医学和组织工程领域的研究人员探索整个血管化器官的生成铺平了道路，真正改变了实体器官移植领域。不要忘记，使用人体器官和组织以及 SDS 等化学品可能极其危险，在进行此程序时，始终佩戴适当的个人防护设备。

Research

JoVE Journal

Bioengineering

Decellularization and Recellularization of Whole Livers

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

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

脱细胞猪心逆行灌注的程序

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

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

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, ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

在振荡压力灌流器程序大鼠肝脏的脱细胞

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

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 ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Isolation and Decellularization of a Whole Porcine Pancreas

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 ...