倒置方向下整个人的心脏在加压袋内的去细胞化
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 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?…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2-0 silk suture | Ethicon | SA85H | Suture used to ligate superior and inferior vena cava |
| 1/4" x 3/8" connector with luer | NovoSci | 332023-000 | Connect aorta and pulmonary artery |
| Masterflex platinum-cured silicone tubing | Cole-Parmer | HV-96410-16 | Tubing to connect heart chambers/veins |
| infusion and outflow line | Smiths Medical | MX452FL | For flowing solutions through the vasculature |
| Polyester pouch (Ampak 400 Series SealPAK Pouches) | Fisher scientific | 01-812-17 | Pericardium-like pouch for containing heart during decellularization |
| Snapware Square-Grip Canister | Snapware | 1022 | 1-liter Container used for perfusing heart |
| Black rubber stoppers | VWR | 59586-162 | To seal the perfusion container |
| Peristaltic pump | Harvard Apparatus | 881003 | To pump fluid through the inflow lines and to drain fluids |
| 2 L aspirator bottle with bottom sidearm | VWR | 89001-532 | For holding solutions/perfusate |
| Quant-iT PicoGreen dsDNA Assay kit | Life Technologies | P7589 | For quantifying dsDNA |
| Calf thymus standard | Sigma | D4522 | DNA standard |
| Blyscan Glycosaminoglycan Assay Kit | Biocolor Ltd | Blyscan #B1000 | GAG assay kit |
| Plate reader | Tecan | Infinite M200 Pro | For analytical assays |
| GE fluoroscopy | General Electric | OEC 9900 Elite | Angiogram |
| Visipaque | GE | 13233575 | Contrast agent |
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Cite This Article
Taylor, D. A., Sampaio, L. C., Cabello, R., Elgalad, A., Parikh, R., Wood, R. P., Myer, K. A., Yeh, A. T., Lee, P. Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation. J. Vis. Exp. (141), e58123, doi:10.3791/58123 (2018).