The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna J&#246;hrens and Katharina Struecker for their help during this project.

动物保持在该基金用于实验医学杂志（FEM，查理特，柏林，德国），并且所有实验方案进行了审查并批准由卫生部和地方事务的德国国办（LAGeSo，柏林;注册号Ø0365 / 11）。 1.肝收获<ol><li>手术前准备<ol><li>使用软木塞板固定。把医用被单上盘。采用四针，一个固定吸入面罩在软木板上术麻醉吸入。 </li></ol></li><li>门静脉插管酝酿<ol><li>一个外周静脉导管（G 20）连接到一个三通阀。静脉导管应斜切，以约的长度。 1.5厘米。 </li><li>去空气使用10毫升注射器填充有Ringer溶液的系统。这是非常重要的是要仔细地去空气，因为气体栓塞可能负面灌注脱细胞的影响。 </li></ol></li><li>酝酿动脉插管<ol><li>使用蝴蝶套管切针5毫米的长度。适合5cm的管（ID0.58毫米; OD0.96毫米）至5毫针刺，用强力胶固定。插入2厘米长的管子（ID0.28毫米; OD0.61毫米）到更大的管和强力胶固定。 </li><li>另一个滑倒在管这种结构桥基和具有强力胶固定。去空气中的动脉插管用5毫升注射器填充有林格氏溶液。 </li></ol></li><li>麻醉与镇痛<ol><li>诱导氧大鼠在麻醉诱导室由3.5％异氟烷的麻醉以1.5升/分钟的流速。 </li><li>为了确保在操作过程中安全镇痛，腹膜内注射安乃近（100毫克/千克体重）和低剂量的氯胺酮/美托咪定（每公斤体重10毫克/ 0.1毫克）。 </li><li>经由吸入面罩用于后续维护外科麻醉的供给1.5％异氟烷在1升/分钟的氧气。 </li></ol></li><li>术前安排&lt;OL&gt; <li>用电动剪毛机剃除宽松的腹部。滋润剃切口部位上，用70％乙醇消毒腹部。 </li><li>将动物在所制备的板仰卧位，插入头插入吸入面罩和修复用医用胶带和标签的肢体。去除多余的乙醇和毛皮用纱布敷。 </li></ol></li><li>麻醉评估<ol><li>通过观察呼吸频率评估麻醉深度。当麻醉似乎足够深（40-60次/ min），检查镇痛是否足以试图通过使用触发钳手术捏两个后肢的脚趾之间的皮肤脚趾捏反射。 </li></ol></li><li>腹途径<ol><li>做一个正中切口入尾腹壁，膀胱上方。从这点切割以在一个V形的方式两个横向肋拱。小心不要切入隔膜。拉剪腹壁在胸前。 </li&gt; <li>修复剑突与overholt钳，颅拉它，并用橡皮筋固定。用湿棉签疏散肠。裹在湿纱布包扎肠道。此外，覆盖所有腹部利润率用湿绷带。 </li></ol></li><li>肝准备<ol><li>解剖镰状韧带。用湿纱布绷带以保持内侧和左肝叶颅下膜片的圆顶。暴露肝十二指肠韧带和上大网膜肝叶。 </li><li>使用两个microforceps和弹簧剪刀仔细解剖附着于上网膜瓣韧带，直到瓣移动。用湿棉签在纱布压缩，保持内侧和左叶的地方移动瓣。 </li><li>动员上部网膜肝叶后，将胃到左侧。修正了一个夹子胃和剖析未成年人网膜与microforceps和弹簧剪刀。 </li><li>动员下网膜升肝叶直到它移动，并随后移动叶回其空腔中。使用巴克豪斯钳保留十二指肠。移动十二指肠向左伸展和暴露肝十二指肠韧带。外接在拉伸肝十二指肠韧带胆总管。 </li><li>解剖来自脂肪和胰腺组织的胆管。切开胆管1.5厘米从胆管分叉。解剖来自周围脂肪组织的门静脉。露出胃静脉。 </li><li>丝绸6-0结扎静脉胃十二指肠两次划分2间结扎静脉。从周围组织解剖腹腔干和肝总动脉，直到所有动脉支显露。 </li><li>解放从周围组织的胃十二指肠动脉。丝绸领带6-0胃十二指肠动脉两次，彼此遥远的联系。解剖2间结扎胃十二指肠动脉。 </li><li>解放脾动脉从周围的组织。丝绸领带6-0脾动脉两次，彼此遥远的联系。解剖2间结扎脾动脉。 </li><li>解放从周围组织的胃左动脉。丝绸领带6-0胃左动脉两次，彼此遥远的联系。解剖2间结扎动脉。 </li><li>外接右肾和右外侧肝叶之间的下腔静脉。从解剖腹膜后间隙静脉。按照腹腔干暴露主动脉。 </li><li>解剖腹膜后脂肪组织。注入1000的IE肝素在1ml盐水进入下腔静脉。收回插管，用化妆棉关闭切口。等待1-2分钟肝素的全身效应。 </li><li>外接钳主动脉。解剖主动脉和抽血的老鼠。此外，从肾脏远侧解剖下腔静脉。 </li></ol></li><li>门静脉插管（使用1.2节准备的套管） <ol><li>使鱼口内切口，在远端门静脉。打开切口，用血管钳和导管插入门静脉。冲洗用20毫升林格氏液，直到肝脱色肝脏和修复连字（6-0丝）的套管。 </li></ol></li><li>动脉插管（使用第1.3节准备的套管） <ol><li>上述解剖腹腔干主动脉。从解放腹膜后间隙主动脉段。切开主动脉段纵向产生主动脉补丁。将自建套管插入静态持有。 </li><li>使用2镊子滑动主动脉修补过套管。结扎肝动脉丝6-0在套管和修复补丁的对接。 </li></ol></li><li>肝外植<ol><li>开隔膜，使一个环形切口，并从周围结构解剖肝脏。 </li></ol></li><li>存储<ol><li>申通快递再在无菌500毫升罐填充用350ml林格溶液直到使用肝脏。 </li></ol></li></ol> 2.洗涤剂溶液<ol><li> 10％的Triton X-100原液<ol><li>填写1,000毫升瓶装900毫升水作空白。并加入100 ml的Triton X-100（99.9％）。使用搅拌器以溶解的Triton X-100。把瓶子装满水作空白。直到1000毫升的总体积为止。 </li></ol></li><li> 1％的Triton X-100的<ol><li>加入100 ml的储备溶液（10％），以1000毫升瓶装。添加900毫升水作空白。并两次摇瓶。过滤通过无菌过滤的1％的Triton X-100溶液。 </li></ol></li><li> 10％的SDS原液<ol><li>填写1,000毫升瓶装900毫升水作空白。并添加66.99克SDS（99.9％，密度0.67）。使用搅拌器以溶解的SDS。把瓶子装满水作空白。直到1000毫升的总体积为止。 </li></ol></li><li> 1％SDS中<ol><li>加入100 ml的储备溶液（10％），以的1000ml的机器人颞叶癫痫。添加900毫升水作空白。并两次摇瓶。过滤通过无菌过滤的1％SDS溶液。 </li></ol></li></ol> 3.脱细胞<ol><li>建立灌流器的<img alt="图1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> 图1:在振荡压力条件下灌注设备的选择性动脉和四大鼠肝脏门静脉灌注的方案 （ 从 Struecker 等人修改14） <ol><li>连结其漏极连接到一个三路活塞和一个海德堡扩展以调节填充水平和填充反应器，用500ml 0.9％NaCl洗涤。 <img alt="图2" src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> 图2:的灌注设置方案链接灌注腔（1,400 毫升 ）（1）到远端的气泡阱（5）经门静脉通路（4）（2）或通过动脉交流塞斯（3）。气泡收集器（5）应配备一个密闭（4）在远端和一通气口（6）。泡沫陷阱（5）连接到海德堡扩展（7）。链接海德堡延伸到泵段（8）。此外，泵段（8）连接到另一个海德堡分机（9）。插入最后海德堡延伸部（9）进入瓶子的洗涤剂溶液（10）。呼吸器（11），以将灌注腔连接（或在多个灌注的情况下，压力分配器）。排出反应器中的流体，连接流出到废液瓶（12）。 <ol><li>插入泵段进入泵内。启动泵以填充循环用PBS，关闭气泡阱的远端和打开排。当气泡的底部被牢固地覆盖，用PBS（2-3厘米），关闭排气口和打开所述气泡阱的前端。 </li><li>肝安装<ol><li>链接门静脉插管的活塞三方的相关反应器的访问。 CONNECT动脉插管动脉进入。要小心，以确保没有空气进入系统。 </li></ol></li><li>的脉动压力条件下的应用<ol><li>使用呼吸器具有15 BPM的频率和26毫巴（最少4毫巴，最大30毫巴）的幅度。连接呼吸器到压力分配室和腔室连接到反应器中。开始施加压力。 </li></ol></li><li>脱细胞协议<ol><li>执行灌注步骤以5毫升/分钟的流速。开始用1％的Triton X-100肝脏进行90分钟的灌注脱细胞。经由灌注装置的流出允许废物流出，以保持内灌注腔常数和灌注肝脏高于液体高度。 90分钟后，将洗涤剂更改为1％的SDS。 <img alt="图3" src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="表1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;P&gt; 图3:所有实验组灌注协议。

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</ol>

The authors have nothing to disclose.

虽然提出了技术大鼠肝收获和脱细胞是容易复制，也有考虑某些关键步骤: 在制备过程中对肝收获，它避免严重出血是非常重要的，因为它会激活血液凝结，并可能导致在肝脏内的血液凝块的形成。在我们看来，这是有利的，门静脉插管之前直接切开腹部大动脉，以避免通过门静脉灌注过程中通过肝动脉血液流入。一旦肝脏插管并明确灌注，血液凝块的形成是不再是一个问题。 后肝收获和运输到灌注设备，肝脏的灌注装置的连接是特别关键的步骤，因为必须避免气泡的进入灌注圆，以防止气体栓塞。它预填的三通阀，是有用的，我s已连接到肝脏插管，使用PBS中的注射器和薄插管直接阀连接到灌注系统之前。 灌注建立之后，是否所有的管接头，三通阀和管连接完美应重新检查。如果肝脏灌注较长时间（ 例如 ，O / N），在连接甚至一个小的误差可导致空气虹吸进入灌注系统，从而导致致命灌注的结果。 所呈现的数据是由以下事实的最佳脱细胞结局振荡压力的变化的最佳压力和频率仍不清楚的限制。此外，压力控制灌注可能导致更好和更稳定的效果比流量控制灌注一样。压力控制灌注使之一灌注协议不同的尺寸和重量，具有相同的结果的肝脏中的应用，因为流会自动bË调整。 肝动脉和门静脉在大小和生理流速方面显著不同。因此，一个恒定灌注速度（5毫升/分钟）必将导致两个血管系统内的不同的灌注压力。类似地，恒定灌注压力会导致不同的灌注速率。一种方法是通过两个途径灌注比较脱细胞的功效将是在通过两种途径生理灌注压或灌注率执行灌注。但是，为了使结果可比在当前的研究中，我们选择了一个恒定的灌注率均为路由。不幸的是，使用我们的设备，我们没能测量灌注肝脏内的灌注压。 我们下一代灌注设备将较小，以减少所需的灌注介质的量。此外，泵装置可使压力控制灌注。 钍Ë提出的技术出现重现性好，均匀大鼠肝脱细胞十分有益。无论是技术，提出适合recellularization和器官体外成熟还需要进一步解决。无论是振荡压力条件对重新填充细胞的影响将是进一步的实验对象。 所提出的技术比其他老鼠肝脏灌注设备更复杂，更显示了几个明显的优势:1）均匀的脱细胞是可重复的，具有良好的效果，当振动压力条件适用。 2）灌注装置是密封的，使无菌脱细胞，这对于矩阵再增殖和长期灌注的先决条件。 3）所提出的协议比其他协议公布短，但还是很有效的。门静脉和肝动脉4）选择性灌注使得通过不同的系统选择性细胞再增殖可能，这可能是一个重要的工具。 灌注装置将在进一步脱细胞实验应用于提炼和优化大鼠肝脏脱细胞。压力控制灌注的效果进行实验评价，并与流量控制灌注比较。加入另外的元素（ 例如 ，一个"透析单元"，一个热交换器，一个充氧器），用于再增殖，培养和成熟实验似乎可行的和合理进一步发展所呈现的设备。

脱细胞和recellularization可以使得能够在体外 1的功能，移植器官的生成。通过除去细胞和抗原物质（ 例如 ，DNA，α-半乳糖表位）从一个器官，非或更少免疫原性外基质（ECM）能够得到。此矩阵可以节省一个器官的三维显微解剖和可作为复育的理想生物基质具有不同的，可能异种来源2的细胞。因此，脱细胞的大鼠肝基质可重新填充与人肝细胞。此人源化微肝脏可以作为离体模型上的疾病（ 如 ，先天性代谢疾病，病毒性疾病或恶性肿瘤）或用于临床前的药物测试3研究。 大鼠肝脏灌注脱细胞几种不同的协议已经公布4-13。在所有协议，脱细胞化是由碱性离子或非离子型去污剂灌注经由插管门静脉实现。尽我们所知，我们是第一批通过选择性灌注经门静脉和/或大鼠肝动脉14报告大鼠肝脱细胞。使不同的血管系统，可在肝脏的选择性灌注可以使更好脱细胞的结果，而且，可以在细胞再增殖中起重要作用。 在这项研究中这里详述，肝脏灌注在一个特制的专用灌流器，振荡压力的条件下，使灌注。这些压力条件模仿肝脏的生理呼吸依赖性灌注: 原位 ，肝脏的隔膜，其变动在呼吸过程中对肝灌注有直接影响的连接函数下挂起。灵感具体地说导致降低隔膜和挤压肝脏，优化肝静脉流出，而到期导致肝脏升高和降低腹内压，优化门静脉流入15。 我们的目的是评估振荡压力条件是否具备通过模仿腹内条件体外对大鼠肝脏灌注脱细胞的同质性产生影响。脱细胞过程的同质性可能是灌注脱细胞低估的因素。所有已知的药物用于肝脱细胞的事业改建为ECM。在灌注不良的地方细胞保持在ECM内，而其它地区已经完全脱细胞。以溶解剩余的细胞中，灌注持续时间或压力必须提高，从而导致更多的改建为良好灌注的区域。因此，洗涤剂脱细胞应均匀器官内分布。

<table><tbody><tr><td colspan="4">Self built arterial cannula</td></tr><tr><td>&amp;nbsp;Portex Non Sterile Polyethene Tubing</td><td>SIMS Portex</td><td>REF 800/110/100</td><td>0.28 mm ID 0.61 mm OD</td></tr><tr><td>&amp;nbsp;Portex Non Sterile Polyethene Tubing</td><td>SIMS Portex</td><td>REF 800/110/200</td><td>0.58 mm ID 0.96 mm OD</td></tr><tr><td>Venodrop Safe butterfly catheter</td><td>Fresenius Kabi</td><td>3275851</td><td>21 G</td></tr><tr><td colspan="4">portal vein cannula</td></tr><tr><td>Periphereal Venous Catheter</td><td>BD</td><td>393224</td><td>BD Venflon Pro 20G</td></tr><tr><td>Three-way stopcock</td><td>smiths medical</td><td>888-101RE</td><td></td></tr><tr><td colspan="4">surgery</td></tr><tr><td>Cotton Sticks</td><td>Hecht-Assistent</td><td>4302</td><td></td></tr><tr><td>Cotton Pads&amp;nbsp;</td><td>Shaoxing Zhengde Surgical dressing</td><td>13H118-03</td><td></td></tr><tr><td>Gauze Bandage</td><td>Hubei Haige&amp;nbsp; Medical Instruments</td><td>14388</td><td></td></tr><tr><td>&amp;nbsp;Ringer Solution</td><td>Fresenius Kabi</td><td>13 HKP022</td><td>1000 ml</td></tr><tr><td>10 ml Syringe</td><td>Braun</td><td>4606108V</td><td>10 ml/ Luer Solo</td></tr><tr><td>5 ml Syringe</td><td>Braun</td><td>4606061V</td><td>5 ml /Luer Solo</td></tr><tr><td>Suture (Silk 6/0)</td><td>Resorba</td><td>H1F</td><td>LOT 105001.81</td></tr><tr><td>medical drape</td><td>Shaoxing Zhengde Surgical dressing</td><td>D0613011</td><td></td></tr><tr><td colspan="4">surgical instruments</td></tr><tr><td>needle holder</td><td>Geuder</td><td>17570</td><td></td></tr><tr><td>micro-forceps</td><td>Inox-Electronic</td><td>91150-20</td><td></td></tr><tr><td>micro-scissors</td><td>Martin</td><td>11-740-11</td><td></td></tr><tr><td>micro-forceps</td><td>S&amp;amp;T&amp;nbsp;</td><td>112314</td><td></td></tr><tr><td>Clamp</td><td>Aesculap</td><td>BH111R</td><td></td></tr><tr><td>scissors</td><td>F S T&amp;nbsp;</td><td>14501-14</td><td></td></tr><tr><td>surgical forceps</td><td>Aesculap</td><td>BD 557</td><td></td></tr><tr><td colspan="4">Decellularisation</td></tr><tr><td>Respirator</td><td>Resmed</td><td>14.24.11.0004</td><td>SmartAIR ST</td></tr><tr><td>Perfusion Device</td><td>Charite, medical engineering laboratory</td><td>custome-made device</td><td>decellularisation device</td></tr><tr><td>peristaltic pump ismatec reglo ICC</td><td>IDEX</td><td>ISM4408&amp;nbsp;</td><td>4-channel</td></tr><tr><td>heidelberger extension 75 cm&amp;nbsp;</td><td>Fresenius Kabi</td><td>2873</td><td>75 cm</td></tr><tr><td>MS/CA pump-segment</td><td>IDEX</td><td>IS 3510&amp;nbsp;</td><td>MS/CA/click&#039;n&#039;go/POM-C</td></tr><tr><td>CA 2-stopper tube</td><td>Pharmed</td><td>BPT NSF-51</td><td></td></tr><tr><td>bubble trap&amp;nbsp;</td><td></td><td>custome-made item</td><td></td></tr><tr><td>Luer Lock hose connector</td><td>Neolab</td><td>No. 02-1887</td><td></td></tr><tr><td colspan="4">Detergents</td></tr><tr><td>SDS pellets</td><td>Carl Roth&amp;nbsp;</td><td>CN30.4&amp;nbsp;</td><td>2.5 kg</td></tr><tr><td>Triton X-100</td><td>Carl Roth&amp;nbsp;</td><td>3051.1&amp;nbsp;</td><td>10 L</td></tr><tr><td>PBS&amp;nbsp;</td><td>Gibco</td><td>14190-094</td><td>DPBS</td></tr><tr><td colspan="4">staining</td></tr><tr><td>Eosin 1%</td><td>Morphisto</td><td>10177</td><td></td></tr><tr><td>Mayer hematoxylin</td><td>AppliChem</td><td>A4840</td><td></td></tr><tr><td>gomori staining</td><td>Morphisto</td><td>11104</td><td></td></tr><tr><td>AlcainBlue-PAS staining</td><td>Morphisto</td><td>11388</td><td></td></tr><tr><td>Direct Red 80&amp;nbsp;</td><td>Sigma Aldrich</td><td>365548</td><td></td></tr></tbody></table>

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.

的均匀性并且因此不同脱细胞协议的有效性是由肉眼观察，组织学分析，和内脱细胞肝基质剩余DNA含量的分析评价。此外，腐蚀铸件进行脱细胞后肝脏可视化的完整的显微解剖。 肉眼在脱细胞，肝脏变得透亮，说明去除细胞含量。肝脏通过门静脉灌注期间和之后脱细胞（白色箭头）呈不均匀澄清（并因此脱细胞），用肉眼可见剩余的细胞。肝脏通过肝动脉灌注表现出更均匀脱细胞过程中，没有明显的残留细胞。如果肝脏灌流的振荡压力的应用条件，没有剩余的细胞是可见的，而不管灌注路径。 <img alt="图4" src="/files/ftp_upload/53029/53029fig4.jpg" /> 图4:大鼠肝脱细胞的无振荡压力条件下的宏观结果 。左:鼠肝经肝动脉脱细胞。肝脏出现透亮，无肉眼可见的剩余细胞。右:肝经门静脉灌注。肝脏出现不均匀脱细胞，用肉眼可见的细胞<img alt="图5" src="/files/ftp_upload/53029/53029fig5.jpg" /> 图5:大鼠肝脱细胞的振荡压力的条件下宏观的结果 。左:鼠肝经肝动脉脱细胞。右:肝经门静脉灌注。这两个肝脏出现透亮，无可见剩余的细胞。 组织学评价的脱细胞基质肝组织学的支持评价ED的肉眼观察结果。肝脏通过肝动脉灌注显示没有剩余的细胞，而不管他们是否振荡压力的条件下灌注。肝脏通过门静脉灌注显示剩余细胞的区域，即使它们被灌注的振荡压力的条件的应用程序。然而，振荡压力条件应用在肝脏经门静脉灌注减少剩余的细胞团块。肝脏的ECM是保守，没有跨所有应用协议可见差异。 <img alt="图6" src="/files/ftp_upload/53029/53029fig6.jpg" /> 图6:H /脱细胞肝矩阵E染色 。 AP:通过肝动脉无振荡压力条件下灌注脱细胞肝基质。 A +警:脱细胞肝基质通过振荡压力条件下的肝动脉灌注。 PA-P:脱细胞肝MATR九，经门静脉无振荡压力条件下灌注。 PA + P:通过振荡压力条件下，门静脉灌注脱细胞肝基质。箭:剩余的细胞团。 DNA含量每肝基质干重的DNA含量各实验组与下降，在本地肝脏相比，虽然差异均有统计学上仅在显著振荡压力条件下（PV + P A + P）下灌注组。每干重的DNA的最低量，发现在肝脏经肝动脉振荡压力的条件下灌注。 <img alt="图7" src="/files/ftp_upload/53029/53029fig7.jpg" /> 图7:肝基质的DNA含量每干重 （从Struecker 等[14]修改）。肝脏通过肝动脉秀脱细胞少剩余的DNA比灌流通过门户网站Ð静脉做。肝脏振荡压力条件下灌注显示比那些没有脱细胞这些条件少做DNA残留。因此，通过肝脏振荡压力条件下的肝动脉灌注显示剩余最少DNA含量。 铸型的脱细胞肝基质腐蚀铸件证实肝脏（包括门静脉系统的蛋白质框架，动脉系统和胆道系统）的显微解剖脱细胞过程中是保守的。 <img alt="图8" src="/files/ftp_upload/53029/53029fig8.jpg" /> 图8:去细胞的大鼠肝基质的腐蚀铸件 （ 从 Struecker 等[14]修改） 的照片 。尽管除去所有的细胞，器官的显微解剖，包括门静脉（蓝色）和动脉（红色）系统，是保守的。该REMA胆道系统都进不去主要分支铸造为黄色。 <img alt="表1" src="/files/ftp_upload/53029/53029table1.jpg" /> 表1:评估的脉动压力条件的影响和路线灌注对大鼠肝脱细胞实验组。

Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

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

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 ...

procedure-for-decellularization-rat-livers-an-oscillating-pressure

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Charité – Universitätsmedizin Berlin. 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.

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

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

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 ...

医学

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

小鼠常母离 体 肝机灌注

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

工程学

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

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

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

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

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

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

生物学

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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 ...

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, ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Evaluation of Apoptosis and Lung Injury in DCD Donor Lungs Preserved by EVLP

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after cardiac death (DCD). However, a critical challenge remains in maximizing the number of viable lungs that meet stringent criteria for clinical use. Ex vivo lung perfusion (EVLP) emerges as a promising technology to evaluate and potentially improve the function of donated lungs. This paper describes the development of a cost-effective and reproducible small animal experimental platform specifically designed for EVLP research. This platform offers a high degree of customization, enabling researchers to tailor experimental parameters to address specific research questions within the field of EVLP. The team established a rat model to simulate DCD and implemented a two-stage preservation approach. Lungs were subjected to static cold storage using a perfusate solution at 4 &#176;C for 4 h, followed by normothermic EVLP for another 4 h. Researchers evaluated various lung function parameters, including pulmonary vascular resistance, pulmonary dynamic compliance, blood gas measurements of the perfusate, and apoptotic cell death in lung tissues. The study demonstrates that EVLP could significantly improve the functional performance of DCD lungs compared to simple cold storage at 4 &#176;C, suggesting that EVLP has the potential to mitigate ischemia-reperfusion injury and enhance graft function following circulatory death, thereby increasing the pool of transplantable lungs.

The objective is to develop a new ex vivo lung perfusion (EVLP) model in rats that can sustain consistent lung function for 4 h throughout EVLP and furnish the industry with implementation specifics that will offer a sturdy method for applying EVLP to rodent models.

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after ...

在振荡压力灌流器程序大鼠肝脏的脱细胞

摘要

探索更多视频

整个肝脏的脱细胞和Recellularization

Subnormothermic的技术

小鼠常母离体肝机灌注

大鼠体内部分肝工程的外科技术基础

大鼠门户成纤维细胞的分离

大鼠肝细胞的分离和小学文化

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

体外常温肝灌注的小动物模型

多参数灌注对照分离原代大鼠肝细胞

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

在振荡压力灌流器程序大鼠肝脏的脱细胞

摘要

探索更多视频

整个肝脏的脱细胞和Recellularization

Subnormothermic的技术

小鼠常母离 体 肝机灌注

大鼠体内部分肝工程的外科技术基础

大鼠门户成纤维细胞的分离

大鼠肝细胞的分离和小学文化

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

体外常温肝灌注的小动物模型

多参数灌注对照分离原代大鼠肝细胞

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

小鼠常母离体肝机灌注

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