Name: a novel surgical technique as a foundation for in vivo partial liver engineering in rat
Uploaded: 2018-10-06T14:00:00
Description: Watch this Scientific Journal Video about A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat at JoVE.com

作者要感谢耶拿大学医院解剖学研究所的 Geiling, 以制作大鼠肝脏解剖示意图。

所执行的住房和所有程序都符合德国动物福利立法。在手术前, 所有纱布、覆盖衣物和手术器械均经过蒸压和准备。所有的程序都是在无菌条件下进行的。
1. 大鼠手术过程的制备
<ol><li>将大鼠放在感应腔内, 麻醉4% 汽化异氟醚, 100% 氧0.5 升/分钟, 直到大鼠完全麻醉。</li>
	<li>把老鼠从感应室取出, 测量它的体重。</li>
	<li>剃掉腹部手术部位的皮毛。</li>
	<li>将动物放回异氟醚腔?...

<ol>
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(92), e51955 (2014).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+and+Recellularization+of+Rat+Livers+With+Hepatocytes+and+Endothelial+Progenitor+Cells.">Decellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.</a> Artificial Organs. 40, E25-E38 (2016).</li><li>Yagi, 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=Human-scale+whole-organ+bioengineering+for+liver+transplantation:+a+regenerative+medicine+approach.">Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.</a> Cell Transplantation. 22, 231-242 (2013).</li><li>Otsuka, H., Sasaki, K., Okimura, S., Nagamura, M., Nakasone, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+co-culture+of+hepatocyte+spheroids+layered+on+non-parenchymal+cells+to+understand+heterotypic+cellular+interactions.">Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions.</a> Science and Technology of Advanced Materials. 14, 065003 (2013).</li><li>Bale, S. 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通过阻断和 cannulating 左门静脉与导管作为流体入口和左外侧肝静脉与另一个导管作为液体出口, 我们成功地产生了体内液体旁路在左外侧叶, 表明虽然该技术是非常具有挑战性的, 由于导管的体积小, 造成出血的高风险, 这是可行的。即使是长时间灌注4小时的老鼠至少存活了1周, 表明大鼠可以忍受这种手术过程。
在下一节中, 我们描述了三技术上最困难和最关键的步骤, ?...

器官移植的适应症不断扩大。相比之下, 器官捐献率和器官整体质量正在下降, 导致对移植物的需求增加。在肝脏移植等待名单中增加的候选人数继续增加 (例如, 在美国, 2016年增加了11340名患者, 与2015年的10636相比)1。尽管作出了大量努力, 但现有器官的数量并不满足临床需要。由于肝病发病率的增加, 许多终末期肝病患者死于移植等待名单, 在捐赠器官成为可利用之前。为满足对供肝移植的巨大需求, 目前正在积极推行采用肝组织工程原理的替代方法2。目前, 一种新研制的肝脏工程生物技术有可能克服这一不足。
肝脏工程由两个步骤组成: 一个脱细胞支架的产生, 其次是支架的重写。为了获得生物脱细胞肝支架, 说明肝通过血管系统灌注离子或非离子洗涤剂, 可以去除肝脏中的细胞材料。在以往的大多数研究中, 用 TritonX100 钠和月桂酸盐联合灌注肝脏, 实现了一种生物脱细胞肝支架。因此, 所有细胞都被移除, 而细胞外基质的结构得以维持。reseeded 的器官支架与成熟细胞、肝细胞以及内皮细胞系和原发肝细胞有或不同时应用内皮细胞或间充质干细胞 (MSC)。大多数研究人员专注于体外肝脏工程3,4,5,6,7,8,9,10, 11,12,13,14。然而, 在以前的研究中, 只有小块的填充支架立方体被移植到不同的异位植入部位。在一些研究中, 部分填充支架移植为辅助移植。然而, 最大报告的生存时间只有72小时8,14。据我们所知, 填充全肝移植的原位移植尚未进行或发表。工程器官的长期功能和移植仍处于起步阶段。因此, 需要另一种方法来进行活体肝脏工程。
在活体肝工程中, 可能是在生理条件下研究肝重写的一种替代方法。与体外肝工程相比, 体内肝脏工程的优越性是多方面的。体外填充部分肝支架采用适当的温度、充足的氧、养分和生长因子与人工培养基进行体外灌注对比, 进行生理血液灌注。此外, 其余部分正常肝脏维持肝功能, 主要允许长期生存。由于植入的体外工程肝移植仍然无法维持实验动物的长期生存, 其肝功能8, 我们设想在体内部分肝 engineeringwould 最终成为一个有前景的模型, 以进一步研究工程肝的进化与更长的生存观察比体内。
最近, 一个研究小组 (潘和同事) 首次提出了在体内肝脏工程的技术15。尽管解剖和技术上的挑战, 他们实现了活体大鼠右下肝叶的分离灌注。他们报告了在体内重写使用大鼠原发性肝细胞系的第一个术中结果。然而,在体内手术灌注模型的 Pan等。有缺点。他们以完全阻断门静脉和下腔静脉为代价, 在大鼠中实现单肝叶灌注, 可能对动物造成严重损害。实验鼠仅在术中观察时间6小时后牺牲。因此,体内肝叶灌注技术需要进一步改善, 才能实现术后生存。
我们开发了一种新的活体肝叶灌注生存模型, 根据前人对16大鼠肝脏解剖的研究, 门静脉插管技术在小鼠17的血流动力学监测和肝脏生物工程18,19. 程序的关键步骤见图 1A - 1E。
该技术适用于那些想使用本实验的活体灌注模型, 用于药物输液部分器官治疗的基础研究,体内去细胞作为器官疾病的化学切除术 (如:, 肝癌),体内细胞培养中的瓣膜基质比较了体外二维和三维细胞培养系统20、21、22、23,24,25,26、在活体肝工程中由去细胞和重写。

<table border="0" cellpadding="0" cellspacing="0" style="width:605px;" width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Perfusion Pump</td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusor VI</td>
			<td colspan="2">B. Braun, Melsungen</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Catheter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versatus-W&#160; Catheter</td>
			<td>Terumo</td>
			<td>SR+DU2419PX</td>
			<td>24G, 0.74&#215;19mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Versatus-W&#160; Catheter</td>
			<td>Terumo</td>
			<td>SR+DU2225PX</td>
			<td>22G, 0.9&#215;25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro surgical instrument</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro scissors</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 14058-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro serrefine</td>
			<td>F&#183;S&#183;L</td>
			<td>No.18055-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro clamps applicator</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 18057-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight micro forceps</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 00632-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved micro forceps</td>
			<td>F&#183;S&#183;L</td>
			<td>No. 00649-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micro needle-holder</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;">No. 12061-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">general surgical instruments</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">standard sissors</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mosquito clamp</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">serrated forcep</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">teethed forcep</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">needle-holder</td>
			<td>F&#183;S&#183;L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">suture</td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 prolene</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 ETHICON*II</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-0 silk</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">11-0 polyamide</td>
			<td style="width:104px;">ethicon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel surgical technique as a foundation for in vivo partial liver engineering in rat

器官工程学是一种新的策略来产生肝脏器官替代品, 可能被用于移植。最近,体内肝脏工程, 包括体内器官去细胞后重写, 已成为一个有希望的方法, 在体外肝工程。然而, 术后生存没有实现。本研究旨在研制一种新的大鼠体内选择性肝叶灌注手术技术, 作为体内肝脏工程的前提条件。我们只通过左外侧叶产生电路旁路。然后左侧叶血气生理盐水灌注。实验以4组 (每组n = 3 只鼠) 为基础, 不同的灌注时间为20分钟、2小时、3小时和 4 h. 生存, 以及在宏观上可见变化的颜色和组织学上确定的血细胞缺乏门户三合会和正弦波, 被作为一个成功的模型建立的指标。左外侧叶选择性灌注后, 我们观察到左外侧叶确实由红色转为淡黄色。在组织学评估中, 在门静脉、中心静脉和正弦波的分支中没有血细胞可见。左外侧叶在重新打开被阻断的血管后变红。12/12 只老鼠在手术中存活了一个多星期。我们是第一个报告一个手术模式的活体单肝叶灌注与长期生存期超过一周。与先前发表的报告相反, 这里提出的技术最重要的优点是在整个过程中保持70% 的肝脏灌注。该技术的建立为大鼠体内部分肝工程提供了基础, 包括去细胞和 recellularization。

十二例男性 (12-13 周) Lewis 大鼠用于评价选择性肝叶灌注的效果。实验以四组 (每组 3只鼠) 进行。使用不同的灌注周期20分钟, 2 小时, 3 小时, 4 小时, 按照上述步骤, 我们成功地在活体单叶灌注。
体内左外侧叶灌注:
血气生理盐水灌注后左...

Watch this Scientific Journal Video about A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat at JoVE.com

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

大鼠体内部分肝工程的外科技术基础

建立了大鼠体内单肝叶灌注模型的新手术技术, 为今后进一步研究活体部分肝工程提供了前提条件。

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

在过去的几年中，肝脏工程已成为全世界的热门话题，因为它具有产生移植能力肝移植的潜力。然而，可行的长期功能和工程器官的移植仍然是未来的愿景。最近，体内肝叶大量移植已成为一个有希望的策略，开始肝脏工程。该技术的一个主要优点是，体内重新填充的部分肝脏支架受到血液分血。这为脚手架提供了适当的温度、足够的氧气、营养和生长因子，与人工培养基的外体富量形成鲜明对比。另一个优点是，剩余的肝脏保持肝功能，因此主要允许长期生存。然而，体内单叶灌注模型在技术上具有挑战性，术后生存尚未实现。在这里，我们将提出另一个手术模型与长期生存作为基础，进一步体内单肝叶工程。在我们演示手术模型之前，我们想简单介绍一下大鼠肝脏解剖。为了建立我们的单肝叶丰富的模型，我们选择了左边叶。这是唯一的肝叶与一个独特的血管供应和排水允许创建一个旁路。这是大鼠门户静脉系统的三维重建。我们在这里的重点是左入口静脉，稍后将作为流体入口。这是大鼠肝静脉系统的三维重建。在这里，我们的重点是左横向肝静脉，稍后将作为流体出口。此肝脏血管解剖图突出了用于部分灌注模型的血管接入点。此动画演示了生成体内左左肝叶灌注模型的方案。为了建立手术模型，我们将在左侧叶内生成一个电路旁路。为此，我们需要阻断以下血管左入口静脉、左肝动脉、左胆管、左中位前静脉、肝动脉和胆管，以及左横向肝静脉。然后，我们需要用24个计具导管来调节左门静脉。之后，使用22个计具导管对左横向肝静脉进行导管。然后，左入口静脉中的导管连接到灌注泵。干纱布被放置在导管出口左侧肝静脉，以吸收废液。这样，将创建旁路。然后左侧叶以每分钟0.5毫升的流速充满肝素盐水。在重新打开堵塞的血管后，左叶的生理灌注恢复。现在建立了手术灌注模型。本视频中演示的所有程序都经过地方当局的评估和批准。腹部的手术场用三轮碘药消毒，然后进行两轮70%酒精的消毒。无菌纱布被放置在腹部，只留下手术场暴露。在老鼠的腹壁上做了一个横向切口。4-0 丙烯缝合线用于修复西腓过程，并将其拉向头部。上腹壁的两侧使用两个亚海岸钩缩回，以达到器官的完全暴露。然后我们可以看到肝叶。这里是用于选择性灌注的左向叶。我们用湿润的纱布覆盖腹腔中的十二指肠和小肠，使卵巢干燥。我们用湿润的纱布抬起中叶，露出肝脏的隆隆。老鼠被放置在无菌显微镜下。我们剖析了左门脉。然后，我们用6-0的丝线缝合将左原静脉缝合。使用微夹，左肝动脉、左胆管以及左中位入口静脉、左中位肝动脉和左中位胆管被阻塞。我们在左横向叶底部用微夹阻断左横向肝静脉。为了创建液体入口，入口静脉的茎被用24个量具针刺破。但是导管没有插入。然后，我们将针头从导管上取出，然后将导管连接到灌注泵。为了将空气从管子和导管中排出，我们用肝素盐水来呼吸一分钟。然后，我们打开泵。接下来，针头无导管，现在连接到灌注泵，插入到左入口静脉。这样，我们最大限度地减少了对可调容器的操纵。这里是左横向肝静脉的暴露区域， 我们将以类似的方式可以。首先，我们用22个量具或24个量具针来打穿左横向肝静脉的排水孔。我们建议导管比容器稍小。然后，我们开始以每分钟 0.5 毫升的流速将左侧叶与肝素盐水进行细足。流出的废液被纱布吸收。为了尽量减少废物液的腹内污染，我们将无针导管插入左侧肝静脉。因此，可曲化的左横向肝静脉作为流体出口。我们继续用肝素盐水来细读左边叶。可以看到血液从出口滴落。箭头表示左向叶在灌注过程中与肝素盐水一起变黄。灌注结束时，导管从容器中取出来。左向肝静脉的排水开口用一个单一的11-0波花石缝合关闭。左入口静脉的入口开口也以 11 - 0 的波杜米特缝合关闭。再灌注从从超前静脉卡瓦的回流开始，去除左横向肝静脉上的夹子。从左肝动脉和胆管以及左中位血管结构中去除夹子后，生理灌注得到进一步恢复。重新打开左入口静脉后实现完全再灌注，颜色更改为深红色表示。左叶的灌注。此图像中的白色箭头表示目标肝叶确实选择性地被注入。这里的白箭显示，剩余的叶，其中代表约70%的肝脏保留生理灌注整个程序。左边叶的再灌注。在这里，红色箭头表示左叶在重新打开堵塞的容器后在生理上重新注入。蓝色箭头显示左中叶持续缺血。选择性地向左叶的注入是使用H E染色进行组织学检查的。图 A 显示，在左边叶中，在入口静脉和正弦的分支中检测出没有血细胞。正如预期的那样，红细胞在肝动脉的分支中可见。在图B中，劣质的考达特叶用作控制。血细胞清晰可见，前静脉和肝动脉的分支以及正弦。图C表明，在左左叶中，中央静脉中看不到血细胞。相比之下，图D表明，在控制中，劣质的卡达特叶血细胞在中央静脉中可见。我们连续12次手术实现了100%一周的存活率。我们比较了我们的体内肝叶灌注模型与潘和同事的模型。潘群和我们组在方法和结果上的主要区别在于：他们选择了右低叶，而我们选择了左边叶作为靶向肝叶;他们阻断了静脉和主门户静脉，导致门户高血压。相比之下，我们维持了70%的肝脏的门户灌注;他们牺牲了大鼠间手术，而我们一周的存活率是100%。我们新颖的手术模型的优点是：它在技术上具有挑战性，但可行;100%的存活率证明了这一点。然而，一个限制是，左中叶持续缺血由于门户静脉堵塞。我国技术的潜在应用包括：利用药物灌注可用于体内部分器官治疗;体内部分器官脱细胞化为化学切除;体内细胞培养系统与体内外细胞培养系统相比;体内肝脏工程。

Research

JoVE Journal

Bioengineering

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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...