从健康的美国国家研究院​​的资助（授予R01EB008678，R01DK096075，R01DK084053，R00DK088962和F32 DK103500），CIMIT项目编号12-1732和施赖纳斯儿童医院的感谢。我们想衷心感谢新英格兰器官银行支持这项工作。

使用人体组织，必须由机构审查委员会（IRB）或同等学历进行审查。这里所描述的工作被批准，并宣布免除由马萨诸塞州总医院的机构审查委员会（第2011P001496）。 1.溶液制备<ol><li>无菌添加补充苯酚如表1所列红的威廉姆斯E培养基。该解决方案应该准备使用前新鲜。胰岛素应在使用前加入。 </li></ol>肝脏2.返回表准备<ol><li>将冰填充的器官碗消毒，披面。从包装盒中取出肝脏，把它留在冷保存液囊中。保持大都淹没肝脏。 </li><li>确定肝动脉（HA），其将位于远端的主动脉补丁。解剖免费动脉揭示各种切枝沿梅岑鲍姆用剪刀长度。仔细狄氏克拉动脉的整个长度，以防止切割供给肝脏的容器中。不要削减或领带没有一个明显的结束分支机构。 </li><li>打结所有动脉分支不供给用丝线缝合范围从大小为0〜4-0，这取决于容器的大小肝脏。关闭分支太短配合或与7-0普理灵一针动脉孔。领带和削减接近其原产地在腹腔干的脾和胃左动脉。 </li><li>通过补丁直接下切腹腔干取出主动脉补丁。导管插入使用主动脉插管腹腔干。 </li><li>识别门静脉（PV）和直截了当地剖析它自由。配合关闭所有分支机构和导管插入PV与大小24管的准备环节。 </li><li>从肝上腔静脉中取出膜片的部分中，不切断静脉本身。流出腔静脉排水沟直接进入器官腔。 </li><li>削减2全圆周TIssue样品（2-3毫米长）从胆总管的结束;卡扣冻结1在液氮中（储存在-80℃）和其他储存在10％缓冲的福尔马林，用于组织和组织学分析，分别。导管插入胆总管与容器插管和一个排放管制成膜式氧合油管。 </li><li>识别并结扎胆囊管与一个0真丝领带。胆囊管的胆总管和胆囊的发现。 </li><li>连接冲洗管设置为冰冷的袋乳酸林格（LR）的解决方案和优质的管材，删除所有的空气。 </li><li>设置冲洗管道的流量调节到一个缓慢滴入。在此之前的冲洗管道连接到门静脉插管，阻塞门静脉用手指在肺门和填充套管和静脉冲洗以从门静脉取出空气。不要提升袋20多厘米了肝脏的高度在寒冷的冲洗，以避免压力过大静脉。 </li><li>在冲洗简要阻塞光伏在最低点。检查PV是否有泄漏。如上所述血管分支可以被关闭。冲洗经PV肝脏总的2升冰冷的LR。 </li><li>医管局与1升LR重复步骤2.10，2.11。 </li></ol> 3.底漆的SNMP系统<ol><li>素加入2升灌注液（21℃）到器官碗和设备开始灌注管道灌注设备。按照该设备的指令，以用于灌注准备，将温度设定为21℃。首先，3毫米汞柱和30毫米汞柱对PV和HA分别压力。打开燃气罐，并设置为3升/分钟的流量。 </li><li>取来自所述HA和光伏流入血液气体样品通过绘制从样品端口0.3 ml样品，并根据制造商的说明在血液气体分析机运行它。确认充分氧合（PO 2&gt; 700毫米汞柱）和pH值（7.35-7.45）。 </li><li>前连接在肝脏取灌洗液的1.0 ml样品截至= 0测量在Eppendorf管中并储存在-80℃。切从使用单刃刀片钢肝脏2±250毫克楔活检;卡扣冻结1在液氮中（储存在-80℃）和其他储存在10％缓冲福尔马林中。灌注前称量肝脏。 </li></ol> 4.人类肝脏灌注<ol><li>肝脏传送到设备。光伏流入连接到PV套管，从PV除去空气如在步骤2.10之后。连接HA以类似的方式。设置的PV和HA压力至3和30毫米汞柱。肝脏应该由灌流几乎被淹没。包括任何干燥的表面，包括流入血管，用湿消毒纱布，防止脱水</li><li>让胆汁管排入收集容器。确保胆汁漏的开口是在肝脏的水平或更低，以允许胆汁运行öUT自如。 </li><li>目标流量分别为275-325毫升/ min.kg和50-100毫升/ min.kg的PV和HA一旦肝脏已经加热到21°C。因为每个肝反应不同灌注，监控在第一分钟内的流动密切。增加或减少的压力在任一船只如果目标流率没有达到。不超过50毫米汞柱的HA和5毫米汞柱的PV。 </li><li>样品可以取自肝脏组织，灌注液和胆汁在研究者的偏好。我们灌注期间建议至少以下样本采集方案。 <ol><li>组织切片中，n = 2×250毫克，每隔一小时。储存:扣在液氮和存储冻结1在-80℃的长期的。此外，采取另一种活检前和灌注后和在10％的缓冲福尔马林固定（N = 1） </li><li>灌注液样品中，n = 2×1毫升，每次15分钟，在第一个小时，此后每隔30分钟。从PV INF借鉴的样本较低的采样口。储存:-80℃长期。 </li><li> PV和HA流入，腔静脉流出血气分析。 N = 3×0.3毫升每30分钟。同时从PV和HA样品端口借鉴样本。通过将注射器插入静脉绘制从腔静脉0.3 ml样品和血液气体分析仪直接运行。使用输出，以保证足够的氧和pH值。 </li><li>胆汁分泌，N = 1×1毫升每一个小时。直观地量化胆汁分泌每隔一小时，并从收集容器的样本。续订取样后的容器。贮存:干冰和-80℃长期。 </li></ol></li><li>继续灌注3小时。监测压力，pH和氧和整个采取样品。通过加入碳酸氢钠至灌流调节他的pH值。 </li><li>在灌注结束走完最后的样本，而肝脏被灌注。断开的肝脏，取出胆管插管。取2后灌注组织样本如前所述储存在-80℃并在10％缓冲的福尔马林胆管。 </li><li>放弃人类肝脏下面妥善处置生物危害的准则。 </li></ol>

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博士。 BE Uygun，K Uygun和Yarmush是发明家在待批的专利，是有关这项研究（WO / 2011/002926），和博士。 BE Uygun，K Uygun和Yarmush是发明家在待批的专利，是有关这项研究（WO /三万五千二百二十三分之二千零一十一）。博士。 ķUygun和Bruinsma有一个临时专利申请与此相关的工作。 ķUygun博士和BE Uygun有经济利益的器官的解决方案，该公司专注于开发器官保存技术。 ķUygun博士和BE Uygun的利益是由MGH和合作伙伴保健按照利益的政策的冲突管理。

在恢复肝脏缺血时受伤的尝试，我们开发了可以在一段冷藏后使用的SNMP系统。 Subnormothermic灌注机提供了一个可行的替代传统的冷库，以及低温和常温灌注机模式。各种不同的系统存在;所有提供不同的优点和缺点3,9,20。 SNMP允许灌注没有氧载体，截至21代谢需氧量 ℃，由灌注液的活性氧满足。 虽然减少subnormothermic条件下，代谢是可观，并且需要的营养丰富的灌注液中的支持。传统灌注液，如贝尔泽机灌注溶液，一般都是最小的组合物和设计用于冷灌注。威廉姆斯的培养基E已被用作肝细胞的培养基多年，并含有Components这是普遍的温暖体外条件下支持细胞功能，尤其如此。 在灌注机的测量结果是反映了器官的功能。直接观察到的参数，如胆汁的生产和氧摄取是可以用于评估肝移植前的实时测量。同样地，细胞损伤和局部缺血（K +，乳酸盐释放）的标记物，可以直接在灌注溶液中测定并可以表示器官功能20。由于灌注机技术的进一步发展，实现更广泛的临床应用， 体外功能和临床结果之间的相关性精确可和灌注参数将在帮助决定移植或拒绝边际品质肝脏有益。此外，如护理分析工具提前点的，更复杂的分析，将机直接期间perfu变得可用锡永21。 在这项工作中，我们表明，肝脏可以在SNMP系统以最小的损伤肝脏支持，通过组织学和ALT的释放反射。肝功能恢复最好是通过ATP的，这已被证明是关联到肝脏的生存能力和强烈提示移植成功的动物模型的反射22。 体外与肝移植物的移植前恢复将使供体的显著扩张肝池，纠正供应和供体肝脏移植需求之间的差距。

肝移植是唯一根治性治疗的谁从终末期肝病患者患有数以万计。为了便于移植成功，从它是从供体，直到时间它被植入在接受者有必要防止移植物的快速恶化采购的时间肝脏最佳保存。目前的标准对于肝脏保存被称为"静态的冷库&#39;:肝脏中冷却的冰冷保存液，从而减少了肝脏的代谢和减缓缺血的有害作用。尽管这种冷藏技术已经允许移植成功，边际质量器官如热缺血或脂肪秀劣质病人受损的器官DCD成果1。有证据一个迅速扩大体肝移植是离体灌注机作为一种替代方式保存可潜在即时结果证明这些边际器官2,3。 肝移植已经成为其自身成功的受害者。远远更多的患者被称为移植比有可用的肝脏，还有数千每年死于等候名单上的美国。由于供肝短缺的现实和次优的质量，有需要的受助人肝移植的提高利用率，人们普遍认为，之前植入的肝脏移植体外灌注机持有肝移植的模式转变的承诺。目前已经在最近几年4-8研究兴趣这个话题在显着增加。在不同的欧洲和北美的中心低温灌注机已取得临床引进8和常温灌注机在生理温度最近已经应用于人类丢弃的肝脏，并正在转化为临床应用，以及9。粗放式发展导致了各种协议的发展，同时不断优化确定最佳的灌注参数10-12。利用边际品质移植增加了超过10倍，在过去十年中13。当与目前的标准对于肝脏保存（静冷库）相比， 离体灌注机提供了许多潜在的好处，所有这些都导致器官池的急需扩张和一个在移植后并发症的发生率可能降低。特别是，胆道并发症移植后，当前困扰次优品质的肝移植仍然是一个重大的问题14-18。 灌注机在subnormothermic条件提供了一个时间窗口，客观地评估肾功能，以适合移植19。而肝脏被灌注在离体电路，无论是灌洗液一个次灌注期间产生的胆汁可取样用于器官功能的标记物的测量值。这样"严重损害"所下的现行标准丢弃移植肝脏可客观评估，以是否适合移植。可行性评估潜在地允许对许多这些器官被用于移植。灌注机的一个同样强大的好处是维修和改进已损坏的暖/冷缺血肝脏。在温暖和随后的冷缺血ATP耗尽非常迅速，可以在之前的20肝脏植入一段灌注机来repleted。肝脏，其能量存储和代谢状态补充，是预处理和再灌注损伤的植入接受者后伤害效果更好的准备。 这项工作描述人类肝脏格拉芙的体外灌注机的方法TS在实验室，这将是研究人员谁愿意学习这两种技术和体外灌注机的有益效果是有用的。我们利用已拒绝移植然后被分配用于研究目的的人类供体肝脏。 标准肝脏采购技术涉及在肝脏下面主动脉阻断脑死亡供体（DBD）或停循环中循环死亡供体（DCD），在其他20多个详细的描述后原位动脉冲洗。另外，肝脏采购通过用冰填充授权人的腹腔中冷却。冲洗溶液偏好区域之间变化，其中大多数使用威斯康星或组氨酸 - 色氨酸酮戊二酸（HTK）溶液大学采购的。门静脉的附加背表冲洗改善的残血的冲刷。肝脏往往采购留下主动脉段河畔舍入腹腔干。胆囊切开，胆汁吸，并胆管被刷新。肝脏被封装在含冰冷的保存液无菌袋，在指定的盒子或冷却器输送。为代表性结果温暖和冷缺血时间应限制在分别为60分钟和12小时，。尽管常规的血清学筛查传染性病原体，标准预防措施必须交给人体器官时，应考虑从人体器官获取样本，以及任何废物。 该协议在这里描述了使用市售的肝灌注装置subnormothermic机灌注。使用这种装置的允许更快速翻译到临床环境和交叉验证之间研究团体和移植中心不同的协议和设备设置的。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Liver Assist perfusion device</td>
			<td>Organ Assist B.V.</td>
			<td>Liver Assist</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Assist disposable set</td>
			<td>Organ Assist B.V.</td>
			<td>Liver Assist disposable</td>
			<td></td>
		</tr>
		<tr>
			<td>Williams&#8217; Medium E</td>
			<td>Sigma</td>
			<td>W1878&#8211;6x500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Eli Lilly &#38; Co</td>
			<td>Humulin R U-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 5,000 U/ml</td>
			<td>Life Technologies</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-156</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>MGH pharmacy</td>
			<td>7750500</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen gas tank 95% O2/5% CO2</td>
			<td>Airgas</td>
			<td>ZO2OX9522000043</td>
			<td></td>
		</tr>
		<tr>
			<td>Specialty gas regulator</td>
			<td>Airgas</td>
			<td>Y11244D580</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated Ringer&#8217;s solution</td>
			<td>Baxter</td>
			<td>2B2324X</td>
			<td></td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Fischer Scientific</td>
			<td>316-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed Adson forceps</td>
			<td>Roboz</td>
			<td>RS-5234</td>
			<td></td>
		</tr>
		<tr>
			<td>Debakey tissue forceps, 7.75&#8221;, 2.25 mm</td>
			<td>Roboz</td>
			<td>RS-7562</td>
			<td></td>
		</tr>
		<tr>
			<td>Metzenbaum Scissors 7&#34; Curved SureCut Tungsten</td>
			<td>Roboz</td>
			<td>RS-6965SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle holder 5.5&#8211;7&#8221;</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>0 blackbraided silk sutures</td>
			<td>Ethicon</td>
			<td>SA66G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 nylon suture, Nurolon RB1</td>
			<td>Ethicon</td>
			<td>C554D</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood gas analysis machine</td>
			<td>Siemens</td>
			<td>RapidPoint 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance scale</td>
			<td>Cole Parmer</td>
			<td>EW-10000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure display box</td>
			<td>Medtronic</td>
			<td>66000</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable pressure display sets</td>
			<td>Medtronic</td>
			<td>61000</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld thermocouple thermometer and probe</td>
			<td>Cole Parmer</td>
			<td colspan="2">EW-91500-04 and EW-08516-55</td>
		</tr>
		<tr>
			<td>Acorn-tipped vessel cannula, 4 mm</td>
			<td>Medtronic</td>
			<td>30005</td>
			<td></td>
		</tr>
		<tr>
			<td>Irrigation set flush tubing</td>
			<td>Hospira</td>
			<td>06543-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixing bowl, 4L</td>
			<td>Cole Parmer</td>
			<td>EW-07300-40</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

可灌注过程中对肝进行了大量的观察和分析，包括直接的实时观测，如流量和胆汁分泌;实时测量，如灌注液的气体分析，并且那些样品采集后由包括灌注液的生化分析和组织学分析事后测量。这里所说的结果是，从22灌注人的肝脏。肝脏被拒绝移植因各种原因，包括捐助者的年龄，过多的热缺血时间，活检结果（脂肪变性，炎症，纤维化）和后勤方面的原因。 18肝脏进行采购的下列心脏死亡和4以下的脑死亡。在这两种情况下，捐赠者进行预处理30,000单位肝素冲洗，并在现场和与UW液表背。平均冷缺血时间为531±237（SD）分和平均热缺血时间为27±10（SD）分，从生活中退出测量支持冷冲洗。时间实际观察和测量可以用来灌注期间评估肝脏，而事后测量灌注后显露出来。 实时观察 通过肝脏流开始比目标流率降低，由于在冷肝脏具有较高电阻的结果。采用3毫米汞柱的PV和30毫米汞柱的HA目标流量一般都可以达到，一旦肝的压力有所回暖至21 &#160; °C 60分钟的灌注（图1A）之后。胆汁流量通常可以内灌注的10分钟观察到的和灌注（图1B）中稳定地生产。胆汁量取决于肝脏的质量，​​并在0.3毫升/小时/千克肝至18毫升/分钟/公斤的范围内。在肝脏较长热缺血时间，胆汁流动将趋向于逐渐减少，而较短的热缺血时间导致更稳定的，甚至增加胆汁分泌。 实时测量 通过血气分析在两种实验目的必不可少的灌注液直接和频繁的测量以及维持适当的灌注条件下，重要的是氧和pH值。溶解氧分压应大于700毫米汞柱两个光伏和HA的流入。流出的氧压力下，在腔静脉测定，通常降低较长灌注，反映一个增加氧量。如先前所描述的13和从0.5-2.2毫升O 2 /分钟/公斤范围在灌注的开始到2.4-9.7毫升O 2 /分钟/千克在t = 3小时（图1C）的氧吸收速率可以被计算。 PH值的下降，在第30分钟（图1D）被观察到的，主要是作为乳酸的释放，在灌注液中的结果。这可以通过补充8.4％钠bicarbo支持内特和大约90分钟后的pH回落到正常范围内。通常，30-50毫升8.4％碳酸氢钠是必需的。乳酸浓度迅速增加，在第15-30分钟，但是开始的第一个小时（图1E）后死亡。 事后测量 肝转氨酶如ALT可以在灌注液进行测定。在第30分钟的大量增加的ALT的通常观察反映的ALT被缺血（图1F）期间释放的冲洗。 ALT被证明与热缺血时间13很好的相关性。灌注机增加ATP含量2.8倍，反映了复苏的能量状态（图1G）。 H＆E组织学分析显示在灌注机（ 图1H，I）持续没有额外的伤害。应当指出，在该协议中提出的活检方案为水库王永立，地球之用，可能并不适用于临床目的。 <img alt="图1" src="/files/ftp_upload/52777/52777fig1.jpg" /> 图1:人类肝脏的过程中机器灌注评价流经PV和HA中的SNMP（A）中，胆汁产生，每小时量化灌注（ 乙 ），氧uprate速率（OUR），从差分中流入计算（光伏+ HA）和流出（腔静脉），间断线表示灌注（℃），pH值和乳酸中的氧分压在入口和流出的灌注期间（D，E），ALT释放到灌注液（F）中，三磷酸腺苷前（H）后（I）灌注从每小时活检肝（54岁DCD，19分钟热缺血，559分钟冷缺血）的（G）和H＆E污渍组织测量的内容。结果表示为平均值±SEM表示。 <aHREF ="htt​​ps://www.jove.com/files/ftp_upload/52777/52777fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion at JoVE.com

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

我们描述了人肝移植在subnormothermic温度（21℃） 的离体灌注机的方法。

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 ...

functional-human-liver-preservation-recovery-means-subnormothermic

Nanyang Technological University

Research

JoVE Journal

Medicine

16.7K 次观看.  Massachusetts General Hospital, Harvard Medical School. 我们描述了人肝移植在subnormothermic温度（21℃） 的离体灌注机的方法。 

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

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

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 liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

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

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

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 ...

科研

医学

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

步骤人类隐静脉<em&gt;前体内</em&gt;灌注和外部强化

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

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 ...

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 ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

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 ...

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, ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

内源性表达人RYR1变异体的功能表征

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Dynamic Navigation in Endodontics: Guided Access Cavity Preparation by Means of a Miniaturized Navigation System

In the case of teeth with pulp canal calcification (PCC) and apical pathology or pulpitis, root canal treatment can be very challenging. PCC are common sequelae of dental trauma but can also occur with stimuli such as caries, bruxism, or after placing a restoration. In order to access the root canal as minimally invasive as possible in case of a necessary root canal treatment, dynamic navigation has recently been introduced in endodontics in addition to static navigation. The use of a dynamic navigation system (DNS) requires pre-operative cone-beam computed tomography (CBCT) imaging and a digital surface scan. If necessary, reference markers must be placed on the teeth before the CBCT scan; with some systems, these can also be planned and created digitally afterward. By means of a stereo camera connected to the planning software, the drill can now be coordinated with the help of reference markers and virtual planning. As a result, the position of the drill can be displayed on the monitor in real-time during preparation in different planes. In addition, the spatial displacement, the angular deviation, and the depth position are also displayed separately. The few commercially available DNS mostly consist of relatively large camera-marker-systems. Here, the DNS contains miniaturized components: a low-weight camera (97 g) mounted on the micromotor of the electric handpiece utilizing a manufacturer-specific connecting mechanism and a small marker (10 mm x 15 mm), which can be easily attached to an individually manufactured intraoral tray. For research purposes, a post-operative CBCT scan can be matched with the pre-operative one, and the volume of tooth structure removed can be calculated by the software. This work aims to present the technique of guided access cavity preparation by means of a miniaturized navigation system from imaging to clinical implementation.

Dynamic navigation systems (DNS) provide real-time visualization and guidance to the operator during endodontic access cavities preparation. The planning of the procedure requires three-dimensional imaging utilizing cone beam computed tomography and surface scans. After the export of the planning data to the DNS, access cavities can be prepared with minimal invasion.

牙髓病学中的动态导航:通过小型化导航系统进行引导通路腔准备

In the case of teeth with pulp canal calcification (PCC) and apical pathology or pulpitis, root canal treatment can be very challenging. PCC are common ...