作者要感谢布罗根，游客米歇尔·韦弗，和Kristen利珀特。资助这项研究是由美国国立卫生研究院资助R03EB009237，以及美国国立卫生研究院的培训资助T32EB001026-06来自国家生物医学成像和生物工程和T32HL076124-05。

1。组织制备及实验设置<ol><li>收获的猪器官后，立即从屠宰场或研究机构的安乐死，并冲洗掉多余的血液。修剪多余的脂肪和组织的心脏，心房和主动脉完整。修剪掉的脂肪分离主动脉肺动脉。如果有任何削减组织，适当地丢弃。 </li><li>单独把每个心，在冷冻纸和存储所有组织在-80°C冷冻至少24小时，以确保完全冻结。 </li><li>当您准备使用（通常不超过3个月），解冻一个完整的冷冻猪心脏在一夜之间淹没在4 L烧杯中的第1类水在4°C </li><li>的心完全解冻后，拍打心脏干，权衡心，并记录重量。市场权重猪的心脏重约375-450克。 </li><li>连接尺寸18的Masterflex管道将带刺的减速和¼“带刺的减速。油管内的主动脉。将2软管夹或主动脉周围的安全拉链关系，只是下面的头臂干。减速器和油管必须保持高于主动脉瓣，所以可以灌注冠状动脉（图1）。 </li><li>使用30或60毫升的注射器，以填补管道与I类水。插入管内的盒的的Masterflex滚子泵，在其近似中点。淹没的流入端的油管在一个4升的烧杯中，用2.5升的水填充的底部和固定管。 </li><li>在装满水的烧杯中，将心和总理的泵，以消除气泡。如果观察到气泡来自管子插入主动脉，主动脉可能需要额外的关系被重新定位或固定。一个密闭的密封是非常重要的，脱细胞过程（图2）的过程中保持足够的压力。 </li><li>将4升的烧​​杯中含有3升的0.2％Trypsin/0.05％EDTA/0.05％NaN的3溶液搅拌板和预热至37°C，在准备的脱细胞处理过程中。 </li></ol> 2。组织冲洗<ol><li>设置泵的流速为400毫升/分钟，确保了正确的油管尺寸被选定。冲洗心脏I类水15-25分钟。由于泵启动时，心脏肿胀和渗出血从心室。新鲜的溶液应被取代的每隔5-10分钟，或根据需要基于从心脏的血液量。如果血液不从心，必要的调整管和夹子三分结构的费托。 </li><li>停止泵和转移心脏充满2X磷酸盐的一个单独的烧杯中，缓冲液（PBS）。后管被浸没在溶液中，启动泵，增加至700毫升/分钟的流速。心应保持在溶液中15分钟，每5分钟改变溶液。每个解决方案的变化需要泵暂时停止，而移动的组织和油管到新的烧杯中。 </li><li>转移心脏I型水10分钟，并增加至750毫升/分钟的流速。 </li></ol> 3。脱细胞和解决方案灌注<ol><li>转移的心脏的烧杯中含有0.2％的Trypsin/0.05％EDTA/0.05％NaN 3的在37°C.泵的转速增加至1,200毫升/分钟，并启动泵。使用搅拌棒的底部放置在烧杯中，烧杯中的溶液循环。的心脏的0.2％Trypsin/0.05％EDTA/0.05％NaN 3的溶液在37℃下应保持在一共有三个小时。 1小时后，增加泵速度为1500毫升/分钟。一小时后，泵的转速增加至1,800毫升/分钟。该组织正在慢慢增加灌注速度条件的组织和防止血管破裂。的心脏会发胀而近一倍的大小，在此步骤中的协议。该组织将失去其自然发展从心房到顶点的颜色，整个日Ë协议（图3）。 </li><li>每个溶液灌注后，一个两步骤进行漂洗，以除去细胞碎片，化学残余物中，援助细胞裂解。每次漂洗由I型由10分钟的冲洗水，接着用2X PBS溶液，在室温下在10分钟的冲洗。每次洗脸的解决方案，从原来的烧杯中去除，加入冲洗液，循环灌流液在烧杯中被淹没的心。 0.2％Trypsin/0.05％EDTA/0.05％NaN 3的溶液后，灌注在1,900 ml / min的水，然后灌注2X PBS中在1950毫升/分钟。 </li><li>转移心3％的Triton X-100/0.05％EDTA/0.05％NaN 3的在室温下的溶液。泵的转速增加至2,000毫升/分钟，一小时灌注溶液。取出从烧杯中的溶液，并用新鲜的溶液替换，增加泵的转速为2100毫升/分钟，和一个额外的一个半小时，灌注的新鲜溶液，使总的时间3％的Triton X-100/0.05％EDTA/0.05％NaN 3的 2.5小时。 </li><li>在2150毫升/分钟和2倍PBS冲洗组织中的I类水2180毫升/分钟，每次10分钟。 </li><li>在室温下4％脱氧胆酸钠溶液转移心脏。泵的转速增加至2,200毫升/分钟和3小时灌注溶液。 </li><li>在I类水冲洗组织和2X PBS在2200毫升/分钟的各15分钟，改变各溶液5-10分钟后的解决方案。所描述的灌注步骤可能被分割多天通过执行两次冲洗步骤和存储心脏附有管，在4℃下过夜，并淹没在I类水。 </li><li>翌日，执行与I类水冲洗5分钟在750毫升/分钟，随后于1500毫升/分钟，用1X PBS冲洗5分钟。然后继续该协议可以在所描述的适当的溶液中的流速。 </li></ol> 4。消毒和最后的处理<ol><li>的心脏转移到0.1％perac的中的客位酸/ 4％的乙醇溶液和1.5小时为2200毫升/分钟的灌注溶液。 </li><li>组织的最终漂洗都执行在2200毫升/分钟。组织1X PBS灌注15分钟，然后用两个5分钟在I类水清洗。重复这一系列的漂洗一次，以完成该溶液灌注过程。 </li><li>将泵关闭，并从溶液中取出心脏排的心。从主动脉切的联系，删除所有的管道，和一个空的烧杯中，将心排1小时。过量的液体将需要定期排出。奠定了心脏的吸水垫，以便完全释放的心（图4）。 </li><li>后，大部分的水被除去，记录心脏的细胞外基质（C-ECM）的重量。心脏可预期的脱细胞过程中失去其初始重量的大约20-25％。 </li><li>解剖左，右心室，以及用于DNA quantificati室间隔和组织学处理，以确认完成脱细胞的组织（图5）。 </li><li>冷冻干燥前的至少2小时，在-80℃下冻结的C-ECM。 </li></ol>

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吉尔伯特博士的科学咨询委员会在ACell，公司正在做的研究，并在最近成为研究和开发的副总裁。 ACell公司销售膀胱矩阵，在本研究中有没有商业利益。

目前的研究描述的一致和有效的方法去细胞的猪的心脏。该协议是一个修改到先前公布的报告，包括更长的曝光时间，流量和压力增加，提供了更多的可重复的结果。由此产生的脱细胞组织满足所有成功的去细胞组织公布的标准。频繁的解决方案进行了修改，以限制重新引入蜂窝材料的组织，每脱细胞处理剂接触到的持续时间减少到最低限度，以减少对ECM的不利影响。的协议的开始阶段期间，灌?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>胰蛋白酶</td><td> Gibco公司</td><td> 15090 </td><td></td></tr><tr><td> EDTA </td><td>费舍尔</td><td> BP120-500 </td><td></td></tr><tr><td> NaN的3 </td><td>西格玛</td><td> S2002-500G </td><td></td></tr><tr><td>的Triton X-100的</td><td>西格玛</td><td> X100-1L </td><td></td></tr><tr><td> 10X PBS </td><td>费舍尔</td><td> BP399-20 </td><td></td></tr><tr><td>脱氧胆酸钠</td><td>西格玛</td><td> D6750-500G </td><td></td></tr><tr><td>过氧乙酸</td><td> Pfaltz和Bauer </td><td> P05020 </td><td> 35％CAS＃79-21-0 </td></tr><tr><td>乙醇</td><td> Pharmco </td><td> 111000200 </td><td></tD&gt; </tr><tr><td>的Masterflex泵驱动器</td><td>科尔帕默</td><td> SI-07524-50 </td><td></td></tr><tr><td>的Masterflex管</td><td>科尔帕默</td><td> 96400-18 </td><td>尺寸18 </td></tr><tr><td>刺减速机</td><td>科尔帕默</td><td> EW-30612-20 </td><td></td></tr><tr><td> 4L烧杯</td><td> Fisher Scientific则</td><td> 02-540T </td><td></td></tr></tbody></table>

procedure for decellularization of porcine heart by retrograde coronary perfusion

灌注的整体器官脱细胞最近获得了为手段，以创建特定的细胞外基质支架，组织工程领域的兴趣，同时很大程度上保留了原生架构的支架。至目前为止，这种方法已经被使用在各种不同的器官系统，包括心脏，肺，肝1-5。没有一个方便的血管网组织往前脱细胞方法都依赖于长时间暴露的组织的解决方案的清洁剂，酸或酶处理，作为一个装置，以除去细胞和核的组件，从周围的细胞外环境6-8。然而，这些方法的铰链的有效性的解决方案的能力后，通过扩散渗透到组织。相比之下，通过自然器官灌注血管系统有效地减少了扩散距离，便利的交通decellularizati代理商进入组织和细胞成分的组织。在这里，我们描述一个的方法完全decellularize通过一个完整的猪心脏冠状动脉逆行灌注。该协议得到充分脱细胞的心肌细胞外基质（ECM）的支架的三维结构体的心脏完好无损。我们的方法，使用了一系列的耦合与高渗和低渗漂洗，以帮助在裂解和去除细胞的酶，洗涤剂，和酸。所使用的协议胰蛋白酶溶液分离后跟的Triton X-100和脱氧胆酸钠的解决方案，以帮助除去多孔材料中的基质细胞。所描述的协议也使用大于2升/分钟的长时间的灌注速度。高流速，加上与解决方案的变化允许运输的代理到组织没有细胞碎片污染和确保有效地冲洗该组织。从National Instruments，简称NI所描述的方法删除了所有核材料维生素E猪心脏组织，创建特定于站点的心脏ECM支架，可用于各种各样的应用。

自然对整个猪心脱细胞的效果的不同而不同的大小的差异，压力，和船只安排。因此，确切的成分派生的细胞外基质支架是不一样的，从心脏到心脏。所描述的协议的完成将产生一个心脏出现白色或半透明状，表明细胞物质的损失。然而，它已被广泛接受的组织可以被视为“脱细胞”的基础上，结合数更多的量化参数8。一个成功的脱细胞协议会产生一个矩阵，每毫克的组织（图6）<...

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

迅速，彻底清除细胞成分通过一个完整的猪心脏逆行灌注的方法。这种方法产生一个站点特定的心脏细胞外基质支架，它有可能用于在多个临床应用。

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 ...

procedure-for-decellularization-porcine-heart-retrograde-coronary

Research

JoVE Journal

Bioengineering

23.7K 次观看.  McGowan Institute for Regenerative Medicine. 迅速，彻底清除细胞成分通过一个完整的猪心脏逆行灌注的方法。这种方法产生一个站点特定的心脏细胞外基质支架，它有可能用于在多个临床应用。

视频: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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

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

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

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 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 ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

通过电容式免疫探 针在猪 心中对神经肽动力学进行时间分辨体内测量

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

在定制灌注生物反应器中获取和灌注-脱细胞猪血管化皮瓣

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

使用 离体 灌注生物反应器进行大鼠后肢的获取和去细胞化血管化复合同种异体移植

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

安装猪主动脉瓣流体动力学特性的评价

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

作者聚焦：在开始大型动物研究之前推进心脏手术测试 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

用于实验性主动脉瓣手术和新型医疗设备流体动力学测试的 离体 猪模型

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...