这项研究由罗氏器官移植研究基金会（ROTRF）和安斯泰来的研究经费支持。马库斯Selzner是由一个ASTS职业发展奖的支持。马蒂亚斯·克纳克由安斯泰来研究奖学金支持。我们感谢乌韦Mummenhoff和伯明翰家族的慷慨支持。

该协议的示意图示于图1。 <img alt="图1" src="/files/ftp_upload/51419/51419fig1highres.jpg" /> 图1研究协议。肝损伤的猪的研究设计基于心脏死亡（DCD）的模式后捐赠。所有的肝血管解剖后，心源性猝死是诱发其次是温暖的移植缺血45分钟。以模拟在临床上的供体和受体的医院之间的移植物运输，移植物后冷，双掣式冲厕储存在冰4小时。冷藏后，该器官是subnormothermic灌流6小时，以评估灌注稳定性。在移植模型中，灌注时间可能会缩短，以充电能量储存和评估机构的生存能力。 <a href="https://www.jove.com/files/ftp_upload/51419/51419fig1large.jpg" target="_blank">请他</a>重新查看本图的放大版本。 1，动物注：男约克夏猪，30〜35公斤，被用于这项研究。所有动物的人文关怀符合&#39;实验动物照护原则&#39;由全国社会医学研究和“指南”对实验动物的照顾&#39;由美国国立卫生研究院公布的制定。在多伦多综合研究所的动物保护委员会批准的所有研究。 2，器官检索<ol><li>宅男约克夏猪的研究设施灌注/移植前1周减少应激水平，并习惯于动物们的居住条件。不到2天壳体里面的设施会导致应力诱导的生理反应，其可以改变灌注的结果22,23。 </li><li>麻醉的猪通过肌肉注射（IM）注射氯胺酮（25毫克/公斤），阿托品（0.04毫克/千克）和咪达唑仑（0.15毫克/千克）的混合物。 </li><li>此前插管，保证猪自然呼吸2升的氧气服用5％异氟醚。插管前喷声带用2％利多卡因2分钟，以避免声带痉挛。例如，对于一个35千克猪用6.5 FR。气管插管。阻塞气管插管用3-5毫升的室内空气。 </li><li>插管后，用capnometry确认无误插管。降低了异氟醚气至2％。呼吸机设置为14-16次/分和10毫升/千克体重的潮气量。 </li><li>放置一个20G的静脉内（IV）导管在耳静脉中的一个，以允许注入的乳酸林格氏溶液（200ml每小时）。然后擦洗猪，需要装无菌单。 </li><li>做中线切口，然后左横向延伸。用毛巾盖住大大小小的肠子，并将它们移动到左侧。 </li><li>独立INFErior腔静脉（IVC）和从彼此远端主动脉;结扎主动脉分支到后面;隔离和贴壁组织游离肾动脉。 </li><li>划分falciforme韧带和使用烧灼三角韧带。 </li><li>通过胰腺和门静脉之间的腹膜切开解除门静脉。扎断静脉胰腺门静脉引流。 </li><li>剖析下面的门静脉腹腔干，并按照它倒退到主动脉。包围肠系膜动脉有2-0平手;围绕脾和胃左动脉，其中分支向后向腹腔干。解剖腹腔干断门静脉。 </li><li>结扎肝十二指肠韧带内的淋巴管，防止淋巴漏。划分关系之间的胃右动脉。结扎小静脉。从韧带分离胆管结扎后远端分裂它。 </li><li>解剖心脏和腹腔TR之间的隔膜背后的主动脉UNK;周围放置主动脉2-0平手。 </li><li>从右侧利用电烙术下静脉释放肝脏;用剪刀对静脉和肝之间的上部。 </li><li>切除胆囊和烧灼从胆囊床出血点。 </li><li>管理肝素静脉千国际单位/公斤体重捐助。对于DCD模式，由心脏内注射40 MVAL氯化钾3分钟肝素注射后诱发心脏骤停。设置心脏停搏作为热缺血的起点。 </li><li>对于灌注，收集心脏死亡后立即1.6升猪血中CPDA袋（柠檬酸盐，磷酸盐，葡萄糖，腺苷）。执行软旋（2000 XG不带刹车）。除去血浆和无菌条件下（生物安全柜II类）下的白膜层和存储在CPDA袋红细胞输血。 </li><li>导管插入门静脉和主动脉器官平齐线。打结围绕股骨，肾，脾，肠系膜和胃左领域的预先设定的关系系列以及上部主动脉。对于心脏跳动的供体（HBD）模型，进行心脏跳动的情况下主动脉和门静脉插管。 </li><li>经过45分钟热缺血，经主动脉（压力袋）和门静脉（重力驱动）采用双灌注冲洗与美国威斯康星大学（UW）解决方案的肝脏。 </li><li>割肝出了猪，让所有剩余的船只长。 </li><li>在后面表制备，使用Satinsky夹紧和逆行通过下腔静脉冲洗肝脏第二次用约0.5升UW液，直到门静脉流出是清楚夹住上IVC。 </li><li>从主动脉和腹腔干系了所有的动脉分支。约0.5升的UW液进行动脉回表的压力灌注。 </li><li>冲洗用UW液胆管。 </li><li>导管插入下腔静脉使用半“×8”减速与鲁尔锁的上部和下部;导管插入用8“门静脉和肝动脉; X 1/4“和6.3”×8“减速带Luer锁。使用上，下腔静脉的静脉引流。 </li><li>将肝脏中的风琴袋，关闭风琴袋，以及肝脏储存在冰直到灌注已经开始。 </li></ol> 3， 体外肝脏灌注<ol><li>制备含有2000毫升斯蒂恩溶液的灌注溶液，加入400ml洗涤红细胞，550毫克丙酮酸钠，加入100ml的氨基酸溶液（10％Travasol），10毫克葡萄糖酸钙，千IE速效胰岛素，将1克头孢唑啉，500毫克灭滴灵， 10,000 IU肝素。添加其它分子的血管扩张，免疫抑制，活性氧的清除，或基于特定研究协议肝脏细胞治疗。 </li><li>对于透析组件，使用标准的透析液含有3.5毫钾，25mM的碳酸氢钠，27 mM的葡萄糖，以及275毫克/升的丙酮酸。 </li><li>成立灌注回路（架构见图2）。 <img alt="图2" src="/files/ftp_upload/51419/51419fig2highres.jpg" /> 图2电路的设置。即将从主储存器的起始和结束点，该灌注溶液通过一个氧合器驱动的离心式泵。该溶液的氧合作用后右，电路分成运行到透析器单元，用于电解质平衡较小的线和更大的线运行到白血球过滤器以减少残存的白血细胞（WBC）的。贯穿透析的溶液返回到主油箱。白细胞过滤后，将电路分割再次成2相等的线。一线路运行在高压力（约60毫米汞柱），直接进入主动脉灌注到肝动脉。其他线路排入第二水库。从这个水库的门静脉灌注。门灌注的压力取决于储层的溶液水平（约2-6毫米汞柱）的高程能量。所有的液体是博士通过基础设施和超肝静脉回主藏ained。为了减少重力均匀灌注，肝脏被放置在装满温度调节水的游泳池。它是从水中分离的防渗膜，它在游泳灌注停牌。 <a href="https://www.jove.com/files/ftp_upload/51419/51419fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <ol><li>收集电路采用3升的储液（主要水库）所有液体和夹紧流出。 </li><li>流出连接到离心泵其次是商业充氧。 </li><li>氧合的背后，分裂成管2行。一条线连接到透析器，并重新流回主油箱。连接第二行白细胞减少过滤器。 </li><li>分手了就行了白细胞降低过滤器插入动脉线，供应灌注液对肝动脉后，和门户v源性行提供的灌注液到第二水库，水渠进入门静脉在重力作用流出。夹紧门户流入。 </li><li>动脉线连接到腔静脉引流线进入主藏流体的回忆。 </li><li>收集腹水或从肝脏灌注溶液的泄漏，准备连接到主储存器的抽吸线。 </li></ol></li><li>松开夹子流出的主要水库，并填写了电路的灌注液。启动离心泵在1500转/分钟。该灌注溶液将通过动脉线路驱动器插入到上腔静脉行返回到主油箱。确保所有的空气赶出了电路。 </li><li>开启气体供给到氧合器。 </li><li>以肝脏落冰。冲洗用生理盐水UW液。 </li><li>将肝，以其凸面朝下，以方便访问到的容器，最好是在重力的环境中，以避免器官压缩时的接触面。用热和冷却的水洗澡。电路和水浴的起始温度设定为20℃。覆盖水浴防渗膜，然后将肝上的膜。浸没灌注液的肝脏减少重力驱动的压缩。 </li><li>减小离心泵的速度到1000转/分，并把两个夹子在动脉和腔静脉线的连接。然后，切管的夹具之间。采用3路连接器，连接两个静脉流出，并将其连接到腔静脉行。 </li><li>释放从动脉线夹紧，倾灌注溶液进入动脉插管除掉气泡，并且线路连接到所述套管。增加离心泵1500转/分钟。释放从腔静脉线的第二夹具。 </li><li>释放门脉水库钳位，以填补它。让灌流液倒入门户cannuLa和连接。要特别注意稳定的流体水平的门户水库。 </li><li>连接压力线的动脉，门静脉和腔静脉插管的鲁尔锁。 </li><li>以模拟生理条件下，应用处理成合适的容器中。注射葡萄糖进入门静脉，而不是进入动脉线，以建立一个模拟的梯度增加门静脉葡萄糖梯度诱导糖原合成24,25。 </li><li>肝脏连接到电路之后，提出在60分钟内温度升至33℃。 </li><li>瞄准动脉起始流量在约250毫升/分钟，在40毫米汞柱。这可能一旦压力增大到70毫米汞柱灌注期间达到700毫升/分钟。 </li><li>在起始温度，旨在为500-600毫升/分钟，在3-5毫米汞柱门静脉的流动。升高温度后，监视门静脉血流量，这将增加高达1100毫升/分钟，在4-6毫米汞柱。避免超过上述生理学门脉压力的L值（约8毫米汞柱），以保护正弦窗孔26。避免超过总流量高于2000mL /分钟，以防止损坏的器官。通过降低主藏，防止肝充血由功能流出道梗阻将流出-2毫米汞柱。 </li><li>透析组分添加到所述电路，以平衡的灌注溶液，以规定的值27。设置透析液流至500毫升/小时。采取特别注意调节透析流出，使得灌注溶液既不稀释也不浓缩。内灌注的第一个小时的主贮存器必须被仔细观看！ </li><li>确保组织的氧合齐用的O 2（95-98％）和CO 2（2-5％）为主的混合气体成分，以恢复并维持器官功能。灌注过程中使用可变气，因为肝脏改变其代谢和灌注过程中pH值的要求。 </li><li>开始时保持低pH值灌注用悖论的pH概念28，以保护器官和避免严重的组织损伤，可能导致从下复氧到生理pH的快速连接中，由于存储在UW液后，将器官具有低于7的酸中毒的pH调节CO的分压2连续下降至25-30毫米汞柱，以使pH值达到1小时内的生理水平。 </li><li>加入钠或碳酸氢钾的电路来实现在灌注溶液标准碳酸氢盐的生理浓度。它注入仔细反复下血气和电解质的控制。 </li><li>监测灌注通过定期静脉和动脉血气和AST分析。静脉PO 2仍高于灌注期间，175毫米汞柱。监控血管的流量和压力，并注意稳定灌流一个常数血管阻力。 </li><li>保持灌注系统稳定长达8小时。在离体灌注期末，凉爽走在灌注系统至20℃，并从肝断开电路的管道后，双重冲洗灌注溶液从肝脏用冰冷的UW溶液。存储再次在冰上放置在无菌的风琴袋肝脏。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+machine+liver+perfusion:+are+we+warming+up.">Extracorporeal machine liver perfusion: are we warming up.</a> Curr Opin Organ Transplant. 17 (2), 143-147 (2012).</li><li>Vogel, T., Brockmann, J. G., Friend, P. J. <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+normothermic+liver+perfusion:+an+update.">Ex-vivo normothermic liver perfusion: an update.</a> Curr Opin Organ Transplant. 15 (2), 167-172 (2010).</li><li>Swindle, M. M., Smith, A. 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R., 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=Liver+transplantation+after+organ+preservation+with+normothermic+extracorporeal+perfusion.">Liver transplantation after organ preservation with normothermic extracorporeal perfusion.</a> Ann Surg. 233 (1), 114-123 (2001).</li><li>Currin, R. T., Gores, G. J., Thurman, R. G., Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+by+acidotic+pH+against+anoxic+cell+killing+in+perfused+rat+liver:+evidence+for+a+pH+paradox.">Protection by acidotic pH against anoxic cell killing in perfused rat liver: evidence for a pH paradox.</a> FASEB J. 5 (2), 207-210 (1991).</li><li>Derveaux, K., 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=Does+ex+vivo+vascular+resistance+reflect+viability+of+non-heart-beating+donor+livers?.">Does ex vivo vascular resistance reflect viability of non-heart-beating donor livers?.</a> Transplant Proc. 37 (1), 338-339 (2005).</li><li>Guarrera, J. V., 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=Hypothermic+Machine+Preservation+of+Human+Liver+Allografts:+Markers+of+Reperfusion+Injury+[abstract#+1282].">Hypothermic Machine Preservation of Human Liver Allografts: Markers of Reperfusion Injury [abstract# 1282].</a> Am J Transpl. 7, 476 (2007).</li><li>Tolboom, 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=Subnormothermic+machine+perfusion+at+both+20&#176;C+and+30&#176;C+recovers+ischemic+rat+livers+for+successful+transplantation.">Subnormothermic machine perfusion at both 20&#176;C and 30&#176;C recovers ischemic rat livers for successful transplantation.</a> J Surg Res. 175 (1), 149-156 (2012).</li><li>Vollmar, B., Menger, M. 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作者什么都没有透露。

在模仿的DCD肝移植动物模型，我们证明了subnormothermic肝脏灌注细胞灌流液产生稳定的灌注参数，最小的肝细胞损伤，激活肝脏代谢。我们subnormothermic灌注成立已经证明来恢复肝细胞内稳态和新陈代谢。糖原贮积恢复和代谢物被丢弃。 体外肝灌注的保护技术提供了在第一次机会器官保存和移植前期间，评估移植物功能和损伤的标记物。旁边的接枝灌注同质的宏观评价，流动数值提供接枝的可行性的良好指标和缺血性损伤，它已受到更早29的程度。耗氧量和胆汁分泌的代谢功能的指标。肝酶样AST的水平可以用来作为SESS肝细胞损伤30度和力度。这彻底的移植评估，可让移植和非移植边缘器官之间的可靠的歧视。 我们选择了33℃的subnormothermic温度中的灌注系统，因为温度足以允许代谢以及ATP和糖原的合成。同时，它提供了一种降低需氧量相比，常温灌注设置，这提供了对缺血性损伤的额外的安全性。在一般情况下，灌注温度高于30℃，已显示，以减少冷缺血性损伤和提供足够的代谢活性31。 相反其它基团，我们不使用全血作为灌注液，但量正常渗透压的白蛋白溶液（斯蒂恩）用水洗，过滤红细胞。通过排除血浆成分以及血小板和白细胞，灌注溶液是去签署，以尽量减少离体灌注过程中的促炎信号。 除了接枝评估，稳定的灌注条件下在几个小时内允许接枝处理。许多分子显示的实验条件下，32来减轻再灌注损伤。然而，几乎没有治疗方案做出了进入临床实践中，还没有。其中一个原因似乎是缺少机会在冷库应用这些治疗方法。在一个体外灌流系统中的代谢活跃的肝脏是最适合应用任何一种治疗。在这点上，不仅治疗以改善再灌注条件等衰减枯否细胞的活性或活性氧的清除都是可以想象的，但也像基因疗法治疗，以调节接枝， 例如 ，针对丙型肝炎复发。其他潜在的战略可能包括体外灌注过程中减少对脂肪肝周期33。 总之， 体外肝脏灌注是一种新的战略，以减少冷缺血性损伤，并评估之前，肝移植边缘供肝。在体外灌注设置提供了独特的机会来修复和移植的条件在移植前。

肝移植是治疗终末期肝病或晚期肝癌的唯一的治疗选择。在过去的25年里，等待名单的候选人人数也逐渐增加，超过了可用移植的数量。心脏跳动捐助者的数量有所减少，在过去的十年。同时，边缘移植物，如心脏死亡（DCD）后捐款，以及老和脂肪肝的数量都增加1,2。 边际移植往往是拒绝，因为原发性移植物无或延迟功能的机会较高肝移植。在DCD移植，缺血性型胆管狭窄（ITBS）的发展是需要特别关注的。与传统的静态低温保存技术，ITBS发生在大约10-40％的DCD移植。在大多数患者中，ITBS导致重新移植或患者死亡。尤其是长时间的温暖和冷缺血时间风险因素ITBS 3-7。供体年龄，遗传倾向（如CCR5增量32），和保存液的选择也已经讨论过的其它危险因素7-10。在胆管周围血管局部微血栓形成已被认为是肝移植术后使用DCD移植物11的潜在机制ITBS。 前的临床引入肝移植的， 体外肝灌注已被用于研究肝代谢和生理学12,13。肝移植术后发现它的方式进入临床设置在20世纪60年代，无数的人们试图用体外肝脏灌注通过模仿生理营养和氧条件下的保存方法。它的实用程序，用于保存边际移植进行了研究，在过去的十年中，但它并没有达到标准的临床护理。最近，我们在D中描述的减少胆管损伤CD肝移植受体外灌流保存14。关于灌流液不同的方法进行。从蜂窝解决方案，如从供体动物全血或浓缩红细胞与血浆结合，脱细胞方法，如威斯康星州的解决方案，IGL解决方案，或斯蒂恩解决方案14-19机器大学的选择范围。 温度范围为4-37℃，20。在低温，subnormothermic和常温的命名是非常可变的和不一致的。所有不同的技术，解决方案，和温度设置旨在1）稳定的灌注条件下，2）足够的氧合作用，和3）重建器官功能。增强的保存容量以及器官评估和治疗的过程中常温和subnormothermic灌注的能力面临更高的技术复杂性，并相比于低温灌注20,21的成本。 我们已经开发了subnormothermic 体外肝脏灌注系统在过去的4年。该系统可用于1）“充电”的肝脏能量含量，2）评估移植物的质量，并以3）修复边缘肝移植前。以下协议中包含的所有信息为稳定的肝脏灌注。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>circuit</td>
			<td>Maquet (Hirrlingen, GER)</td>
			<td>custom made</td>
			<td>main reservoir (3L, 3/8&#34; outflow)</td>
		</tr>
		<tr>
			<td>-</td>
			<td></td>
			<td></td>
			<td>portal reservoir (1.5L, 1/4&#34;, outflow)</td>
		</tr>
		<tr>
			<td>-</td>
			<td></td>
			<td></td>
			<td>centrifugal pump</td>
		</tr>
		<tr>
			<td>-</td>
			<td></td>
			<td></td>
			<td>oxygenator</td>
		</tr>
		<tr>
			<td>-</td>
			<td></td>
			<td></td>
			<td>leukocyte filter</td>
		</tr>
		<tr>
			<td>tubing (1/4&#34; x 1/16&#34;)</td>
			<td>Raumedic (Helmbrechts, GER)</td>
			<td>MED7506</td>
			<td></td>
		</tr>
		<tr>
			<td>tubing (3/8&#34; x 3/32&#34;)</td>
			<td>Raumedic (Helmbrechts, GER)</td>
			<td>MED7536</td>
			<td></td>
		</tr>
		<tr>
			<td>tubing connectors</td>
			<td>Raumedic (Helmbrechts, GER)</td>
			<td></td>
			<td>various sizes</td>
		</tr>
		<tr>
			<td>dialysis filter, Optiflux F160NR</td>
			<td>Fresenius Medical Care (Waltham, MA)</td>
			<td>F160NR</td>
			<td></td>
		</tr>
		<tr>
			<td>STEEN solution</td>
			<td>XVIVO (G&#246;teborg, SWE)</td>
			<td>19004</td>
			<td>2L</td>
		</tr>
		<tr>
			<td>dialysis acid concentrate A</td>
			<td>Baxter (Mississauga, ON)</td>
			<td>D12188M</td>
			<td>45ml</td>
		</tr>
		<tr>
			<td>amino acid, Travasol 10%</td>
			<td>Baxter (Mississauga, ON)</td>
			<td>JB6760</td>
			<td>100ml</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma-Aldrich (St. Louis, MO)</td>
			<td>P2256</td>
			<td>1.1g</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sandoz Canada Inc (Toronto, ON)</td>
			<td>10750</td>
			<td>40000 iU</td>
		</tr>
		<tr>
			<td>Calcium Gluconate</td>
			<td>Pharmaceutical Partners of Canada (Richmond Hill, ON)</td>
			<td>C31110</td>
			<td>10mg</td>
		</tr>
		<tr>
			<td>fast acting Insulin</td>
			<td>various vendors</td>
			<td></td>
			<td>1000 iU</td>
		</tr>
		<tr>
			<td>Cefazoline</td>
			<td>various vendors</td>
			<td></td>
			<td>1g</td>
		</tr>
		<tr>
			<td>Metronidazole</td>
			<td>Baxter (Mississauga, ON)</td>
			<td>JB3401</td>
			<td>500mg</td>
		</tr>
	</tbody>
</table>

technique of subnormothermic ex vivo liver perfusion for the storage, assessment, and repair of marginal liver grafts

肝移植的成功导致了戏剧性的器官短缺。在大多数地区移植的病人轮候肝脏移植的20〜30％死于不接受器官移植或摘牌是疾病进展。一种策略，以增加捐助池边缘移植，如脂肪肝，从旧的捐赠者移植，或心脏死亡（DCD）后，捐赠的利用率。冷静态存储当前的保鲜技术是通过产生显著器官损伤肝脏边缘的耐受性很差只。此外，冷静态器官存储不允许接枝评估或移植前修复。 冷静态保存这些缺陷引发兴趣的温暖灌注器官保存，以减少冷缺血损伤，保存过程中评估肝移植，并探索机会移植前修复边际肝脏。最佳PRES肯定和流量条件下，灌注温度，灌注液的组成和需要的氧载体一直存有争议的过去。 尽管有希望的结果在几个动物研究中，复杂性和成本，防止了更广泛的临床应用至今。近年来，随着技术的提高和体外灌注过程中更好地了解肝脏生理的暖肝血流灌注的结果有所改善，并持续了良好的效果得以实现。 本文将提供有关肝检索，存储技术，并在猪隔离肝脏灌注的信息。我们将示出）的要求，以确保足够的氧供应给器官，b）约灌注机和灌注溶液，和c）生化分离的器官的各方面技术方面的考虑。

下面，我们提出的5灌注实验与DCD-移植后的结果45分钟变暖和4小时冷缺血的subnormothermic 离体灌注的开始之前。 对于离体肝脏灌注的主要目标是确保有足够的氧气供应的器官。缺血引起的血管收缩，从而增加了灌注阻力。实现稳定的压力不断的血管流动是充分的氧合一个很好的指标。在1-2小时的诱导期的灌注溶液和器官被升温至33℃，这deceases肝脏的血管阻力。一旦33℃的目标温度来实现，流量值电平恒定，几乎生理范围的6小时的灌注时间（ 图3A-3D）的其余部分。 同时，在器官变得代谢活性。 图4A示出了静脉PO &lt;子&gt; 2，耗氧量的指标。在最初的2小时静脉PO 2下降至一个恒定的平台。在此代谢活性的状态下，肝脏开始制造胆汁（ 图4B）。透析器提供了一个平衡的电解质平衡（ 图4C-4D）。初始高钾血症迅速拉平。在线助攻测量作为监测肝细胞损伤。 图5显示了只有在整个灌注期浅线性AST升高。 6小时灌注后的H＆E染色显示肝细胞坏死&lt;5％具有完整的小叶和正弦结构（ 图6）。 PAS染色在同一时间点相比，耗尽存储在冷保藏DCD-移植物（ 图7）示出了补充细胞糖原储存。 <img alt="图3" src="/files/ftp_upload/51419/51419fig3highres.jpg" /> 图3：灌注流量和压力（N = 5，误差条显示标准偏差）。 （A，B）肝动脉（HA）的流量和压力：在第一1-2小时期间升温阶段，在稳定压力下的流量的增加而恒定之后。望着降低门静脉压（C）所示 ，HA向灌注的端流的增加可能是肝脏的自身调节反应（C，D）的过程中对应于HA流中的门静脉（PV）的流量增加第2小时升温。压力保持相对稳定。 <img alt="图4" src="/files/ftp_upload/51419/51419fig4highres.jpg" /> 图4监控参数组（n = 5，误差条显示标准偏差）。 （A）中的静脉PO 2作为需氧量和代谢活性的标记物变暖的初始阶段内减少因活化cellulaŘ新陈代谢;它保持稳定之后（B）的胆汁产生代谢活性的标记开始处的温度在30℃左右，因此，灌注的第一和第二小时之间（C，D）的透析器，保证电解质平衡。;最初的高钾血症是迅速平衡。 <img alt="图5" src="/files/ftp_upload/51419/51419fig5highres.jpg" /> 图5助攻（N = 5，误差线显示标准偏差）的AST是肝细胞损伤的敏感指标。浅上升表明体外灌注过程中没有显著伤害。 <img alt="图6" src="/files/ftp_upload/51419/51419fig6highres.jpg" /> 图6的H＆E染色（放大20倍）。热缺血前（A）深的肝脏样本中，一位代表肝小叶与完整architectur后ë（B）肝样品45分钟暖缺血损伤，4小时冷缺血，和6小时subnormothermic灌注，小叶结构是完好无坏死和只有很少的细胞肿胀，正弦空间轻度扩张相比于假样本。

Watch this Scientific Journal Video about Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts at JoVE.com

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

边际移植物，如脂肪肝，从较旧的捐赠者移植物，或心源性死亡（DCD）后取出肝脏耐受常规，冷静态存储仅很差。我们开发了一种新的subnormothermic 体外肝脏灌注保存边际移植肝移植前，评估和修复模型。

Subnormothermic的技术<em&gt;前体内</em&gt;肝脏灌注的存储，评估和修复边际移植肝

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

technique-subnormothermic-ex-vivo-liver-perfusion-for-storage

Research

JoVE Journal

Medicine

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

14.6K 次观看.  Toronto General Hospital. 边际移植物，如脂肪肝，从较旧的捐赠者移植物，或心源性死亡（DCD）后取出肝脏耐受常规，冷静态存储仅很差。我们开发了一种新的subnormothermic 体外肝脏灌注保存边际移植肝移植前，评估和修复模型。 

视频: Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

科研

医学

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

隔离肝脏灌注作为治疗葡萄膜黑色素瘤肝转移

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

供肝易地常温灌注机

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

功能人类肝脏保存和恢复由Subnormothermic机灌注手段

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

常温<em&gt;离体</em&gt;肾脏的灌注肾的移植保护移植前

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

大动物模型肝组织血浆凝固的评价

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. 
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. 
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.

非原生离体胰腺灌注用于移植前胰腺同种异 体 移植物的保存

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ ...