由NIH /的聂EY01​​7885（RMS）的部分支持。

1。设置：无菌技术11 <ol><li>打开层流罩（LFH）。 </li><li>打开氮气控制单元。确保软管控制台单元和氮气罐之间的连接。打开气。设置应设置燃气50-60 PSI稳压罐。汽轮机压力单位应改为3.3至3.4条。 </li><li>按测试按钮。大气压力会读在737-700和泵1和2应该是低于200。单位测试完成时，会发出蜂鸣声。唐面具保护眼睛，如适用，并盖。洗手和唐非无菌手套。 LFH和开放的包裹内放置无菌包，建立无菌区。 </li><li>将消毒药杯和一个既定的无菌场旁边的无菌纱布，无菌塑料脸盆。药杯倒入无菌异丙醇。用酒精棉球/垫和地方探头尖到药杯，测厚仪探头。探针尖端应为10 m的浸泡在异丙醇inutes。测厚仪探头浸泡10分钟后，从预充式注射器无菌水冲洗探头尖端去除酒精和地方探头在塑料盆。 </li><li>使用无菌技术，打开拖放到无菌区无菌物品（叶片，无菌手套，和角膜的观察室），除非已预制包的一部分。 </li><li>在保护媒体和内的LFH地方（ 图2）获取的角膜组织。 </li><li>打开小瓶（S）和生物危害到一个合适的插座盖（S）的处置。丢弃非无菌手套。 </li><li>戴上无菌手套。将预先包装的塑料盆地位于测厚仪领域的一面。输液线连接活塞和地方穗输液线年底油烟机的安全线，在这样一种方式，以防止外界的引擎盖线，从重返引擎盖和接触无菌领域之外。 </li><li>执行角膜组织转移。轻轻倒入组织和媒体，在SIngle议案，从现有的小瓶一个无菌观看室。务必保持血管内皮的一面向上。放置帽室。含无菌区的组织和媒体保持商会。将LFH小瓶（S）。取下并丢弃无菌手套。 </li><li>穗平衡盐溶液（BSS）瓶输液线。挂瓶的无菌的BSS约四个工作区域表面英尺。 </li><li>打开两对无菌手套和消毒用毛巾袍。 </li><li>执行六分钟的手术擦洗和干手器。 </li><li>放在无菌袍，然后双方对无菌手套。 </li><li>到无菌的领域，打开仪器箱和地方的内容。 </li><li>成金属盆或药杯免除10-20 CC BSS解决方案。无菌吸管填充BSS解决方案。活塞和输液线注入前房端口连接和与BSS冲洗，使少量活塞顶部可见。 </li><li>在LFH位置人工室返回纱布。降低活塞移植紧缩环逆时针转动。 </li></ol> 2。程序<ol><li>使用镊子，轻轻抓住角膜巩膜轮辋。打开活塞冲洗BSS系统的同时，认真围绕角膜内皮方活塞顶部下来。如果气泡下方的角膜可见，继续与BSS注入及角膜轻微移动，删除它们。气泡一旦被删除，停止输液。确保角膜中心活塞（ 图3a）。 </li><li>前房盖基地锁紧环的位置，使基地空地盖适合的标签。锁定转化基地盖盖顺时针旋转15°转动。提高活塞和完全拧紧（ 图3b）。 </li><li>用无菌手套的手指，轻触角膜上，以确保有足够的压力来执行程序。 </li><li>检查表面O探头放在测厚仪f角膜获得厚度读数（ 图4）。标准偏差必须小于10。如果没有，重新夺回测量。随着无菌手套的手，没有被用来执行测厚仪，通过涡轮软管手进行测厚仪和涡轮软管拧到控制单元。有协助技术员纪录测厚仪阅读。 </li><li>脱去污染的手套。 </li><li>使用无菌标记笔，画出向角膜中心（或不标记，如果医生喜欢没有标记）从4毫米缘线。使用吸管放置在角膜上五滴的BSS。 </li><li>除非是由医生提供的具体说明，预先切开的角膜厚度测量，如果是小于600微米，300微米切割头将。如果超过600微米，350微米切割头将被使用。 </li><li>对你和数量朝上面临与线程，加载到角膜头部右侧的刀片，首先指出年底，避免接触无线网络TH的刀片。 </li><li>持头，每边拧到涡轮头。人工室用手指拧紧。 </li><li>涡轮软管拧到涡轮机。 </li><li>测试真空踏板一次，按5秒，淹没在无菌水切割头和脉冲涡轮叶片振荡。 </li><li>重新拧紧活塞。 </li><li>安装后，并在五点钟的位置上，基地的地方角膜。按住涡轮踏板振荡刀片（ 图5a）。 </li><li>用食指和拇指，保持角膜之间的中间基地和涡轮机的顶部。转动手腕使平稳，甚至穿过整个角膜。推出涡轮踏板和直客单位解除角膜。按真空踏板，关闭真空（ 图5b）。 </li><li>戴上无菌手套和复检后角膜帽测厚仪阅读被删除放置在角膜表面的探头获得第二厚度读数注意到，该组织是非常薄，很容易损坏。脱掉污染的手套。有协助技术员纪录第二测厚仪阅读。 </li><li>使用无菌记号笔，放置一个标志，如果按医生的要求，在人工腔残余基质床中旬外围边缘。残余基质床放置到无菌的BSS几滴。 </li><li>删除从角膜前角膜帽（ 图6），并取代人工前房角膜在角膜上，然后删除它。第应集中在角膜周边标志着以正确对齐帽，如果适用。使用拭子矛轻轻地定位在角膜上的帽。使用新鲜的矛吸收前盖下方任何的BSS。 </li><li>打开活塞，转腔倒够慢下活塞，以防止变形角膜。解锁基地，并允许从BSS流量的压力，以推动基地的人工腔杯。用钳子，轻轻拉T的角膜在9轮辋，12日和3点钟位置，松开杯。一旦松动，在12点钟位置使用镊子取出角膜。观看室，内 ​​皮朝上（ 图7）放置在角膜。放置在观看室的盖子。取出无菌手套。 </li><li>观看室盖拧紧。 </li><li>执行后切裂隙灯角膜和记录结果的评价和反射镜。 </li><li>地方商会（S）在适当的冰箱节。 </li><li>以外科医生为运输包装（ 图8）。 </li></ol> 3。代表结果适当的机械角膜光滑，均匀的层状角膜供体组织捐献角膜的结果清扫。这是光学相干断层扫描（OCT）图像截面的角膜（ 图9）所示。最后后的角膜组织，应该是足够的厚度由pachymet测量RY。在密歇根州的眼库在过去6个月，平均处理前角膜厚度为558微米，后处理后的角膜组织的厚度为158微米。 87％的100-200微米之间，10.9％计算&gt; 200微米，2.1％计算&lt;100微米的角膜，解剖捐助测量组织。 后的角膜组织，必须保留内皮细胞高密度（ECD）提供改善血管内皮功能的移植。反射镜是用来衡量幼儿。在过去6个月内，所有被视为资格来自密歇根州的眼库内皮角膜移植术的角膜组织幼儿&gt; 2200细胞/毫米2。九十九％的预切DSAEK组织后处理幼儿&gt; 2400细胞/毫米2，和19％的幼儿&gt; 3000细胞/ mm 2。在预先处理后幼儿的平均变化是微不足道的1.2％（P = 0.0003，配对双尾t检​​验）。在手术过程中可能发生的并发症包括亏损的压力，非对称夹层，和显着的组织压痕造成角膜内皮细胞丢失。不当的夹层也可以导致组织穿孔。 <img alt="图1" src="/files/ftp_upload/3847/3847fig1.jpg" /> 图1。内皮角膜移植术图1a至1E角膜组织制备的总体方案截面表示捐献角膜的准备。 图1F 1I DSAEK手术移植的角膜截面交涉。 <ol class="lalpha"><li>捐献角膜是安装在一个人工前房解剖角膜头。 </li><li>组织剥离后，两片层是独立的，但前薄板（帽）的位置上保持角膜床运输医生。 </li><li>在手术室，外科医生使用环钻角膜组织中央垂直穿过前部和后部的鳞片。 </li><li>周边捐献角膜环钻切割后，从外地被删除。 </li><li>后瓣前瓣用锅铲轻轻取出。捐助后瓣（DSAEK组织）取代收件人的角膜病变（1I）的一部分。 </li><li>病人患病后角膜被删除。 </li><li>病后角膜人工仪器在一个大直径的圆（反向Sinsky钩）与取得。 </li><li>进球后的角膜病变，角膜后膜被删除。 </li><li>捐助后瓣（DSAEK组织）被放置在收件人的眼睛。 </li></ol><img alt="图2" src="/files/ftp_upload/3847/3847fig2.jpg" /> 图2。在媒体的角膜组织。角膜组织后，房委会在保护媒体rvesting来自捐助国。 <img alt="图3" src="/files/ftp_upload/3847/3847fig3.jpg" /> 图3。前房人工角膜组织。 <ol class="lalpha"><li>角膜组织被装上人工前房事先基地锁紧环或组织清扫。 </li><li>锁定基地组织在密封环安装到人工前房角膜组织。 </li></ol><img alt="图4" src="/files/ftp_upload/3847/3847fig4.jpg" /> 图4。角膜组织厚度检查。检查角膜厚度，角膜表面上使用超声波测厚仪探头。 <img alt="图5" src="/files/ftp_upload/3847/3847fig5.jpg" /> 图5。角膜组织解剖。 <ol class="lalpha"><li>角膜刀片的基础上安装后，并在五点钟的位置。 </li><li>角膜通过角膜层状剥离。 </li></ol><img alt="图6" src="/files/ftp_upload/3847/3847fig6.jpg" /> 图6。解剖后的角膜。前角膜帽是免费的角膜剥离后的前盖是完整的。 <img alt="图7" src="/files/ftp_upload/3847/3847fig7.jpg" /> 图7。解剖查看商会角膜剥离完成后，角膜返回运输观看室。 <img alt="图8" src="/files/ftp_upload/3847/3847fig8.jpg" /> 图8。在运输包装的角膜。在观看室的角膜，然后安全地包装运输的外科医生。 <img alt="图9" src="/files/ftp_upload/3847/3847fig9.jpg" /> 图9。华侨城华侨城外观角膜。最后解剖角膜（截面）显示，角膜被分裂成两部分。接口是更强烈的反射比周围组织，如箭头所示。

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precut+tissue+for+Descemet+stripping+automated+endothelial+keratoplasty.">Precut tissue for Descemet stripping automated endothelial keratoplasty.</a> Cornea. 27, 630-631 (2008).</li></ol>

博士。伍德沃德和Shtein没有透露。中西部眼银行员工，先生Mavin泰特斯先生。

最里面层，角膜内皮细胞，维持角膜脱水，保持角膜的光学清晰度。许多疾病状态，特别包括营养不良，感染，炎症过程和退化，影响角膜内皮细胞的健康和活力。直到最近，角膜移植，以取代病变的角膜内皮细胞所需的全层角膜移植。在过去十年中，有针对性地更换后的角膜偏厚的角膜移植，DSAEK手术，已经能够用更短的恢复时间，以达到同样的目标。无论技术，移植的组织必须保持一个健康的角膜内皮细胞移植角膜的光学清晰度保持作为一个结果，所有的组织处理技术，具有额外的步骤，以保护角膜内皮。 自动角膜剥离是目前日e标准内皮角膜移植术的角膜组织准备。使用的角膜，以减少捐助组织穿孔，加快视力恢复相比手动清扫5。 DSAEK最初介绍，一些医生购买角膜设备和手术室在手术开始进行供体层状剥离。如果组织准备失败，手术将被取消造成不便和费用的病人和医生。眼库在2006年开始执行角膜的解剖“场外”的前一天手术12眼库实验室的受控条件下。如果在此设置的组织准备失败，有时间来执行另一个新鲜组织解剖。 捐助后板层角膜组织角膜剥离需要温柔和精确的技术，以尽量减少并发症。必须放置在角膜gentlY对人工前房（AAC），以避免任何微妙的内皮细胞的损伤。一旦角膜安装和对齐的AAC，必须定位和锁紧环，一个密封室内保持高的压力和减少组织崩溃的机会，在角膜剥离调整。以确保统一清扫，角膜头应该是一个常数，顺利地旋转。组织剥离后，必须再次进行审慎处理，以减少内皮细胞的损伤。此外，它是至关重要的确认，以确保后的鳞片将是适合于使用在手术13层状切割中心和完整。 角膜捐赠穿孔此过程是可怕的并发症，它用丰富的经验变得越来越频繁。如果反复穿孔发生在角膜的推移，它是重要的检查过程中的多个步骤。玉米EA必须正确安装和对准的AAC。应事先获得一个密封和高压到角膜通，并保持整个过程。有时，存在残余结膜巩膜缘和需要安装前被解剖。角膜刀片应锋利，无缺口。估计角膜厚度的基础上，应选择正确角膜头厚度。角膜头必须正确振荡前通。随着这一技术的进一步问题，咨询角膜的供应商，以确认所有设备工作正常。 即使完全执行时，也有一些这种技术的局限性。角膜头可切割不同厚度的角膜组织，然而，提供广泛的最终组织厚度，角膜头，最后组织厚度的关系是同一头不精确7。此外，在准备一个重要的学习曲线exists和并发症的新用户更频繁。在眼库设置执行的层状剥离，允许执行此过程，每天多次训练有素的技术人员，以优化他们的技术，从而最大限度地减少并发症，由于缺乏经验。 辅助角膜角膜内皮角膜移植存在夹层的技术修改。有些人喜欢使用上提供增强保护角膜内皮细胞的意图前房人工角膜按钮下方的粘弹性材料或角膜的存储介质，而不是平衡盐溶液。在角膜切口应发起位置的确定方法也有变化。不同眼的银行和不同的角膜外科医生可能有特别的喜好，如何识别标记角膜角膜前盖或后板层的方向。应当指出，制造商在本协议所使用的类似设备。无论使用哪种设备，应遵循的特定制造商的指示。 解剖DSAEK手术的角膜组织的银行准备可靠的眼睛一直有利于这个相对较新的手术技术的广泛接受。一旦医生成为眼银行执行的“第一步”，在角膜移植中的信心 - 即组织准备，许多实际的限制DSAEK得到缓解。眼库已经能够可靠地保持与捐助组织供应安全，有效，及时的处理与组织的需求。高品质DSAEK组织现在可以责令提前和手术室的时间和组织准备并发症减少9，14-15。 本协议描述的preparati从眼睛的角膜供体组织的银行处理的角度DSAEK移植;然而，这种技术是直接适用于外科医生在准备自己的角膜板层捐助组织以及感兴趣的兴趣角膜板层上执行基于实验室或临床研究移植。 角膜板层准备未来的发展方向，包括组织处理，利用飞秒激光创造“超薄”DSAEK角膜组织，或准备弹力膜内皮角膜移植术（DMEK）手术治疗角膜内皮滚动。内皮角膜移植术是在进化过程中的手术视野和眼库和角膜外科医生之间的共生关系，将继续改变外科技术进行了优化。

<table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>角膜</td><td>莫里亚</td><td> 1021174 </td></tr><tr><td>人工前房基础的</td><td>莫里亚</td><td> 19161-562 </td></tr><tr><td>人工前房帽的</td><td>莫里亚</td><td> 19172-220 </td></tr><tr><td>角膜总目300 </td><td>莫里亚</td><td> O304 </td></tr><tr><td>角膜总目350 </td><td>莫里亚</td><td> N791 </td></tr><tr><td>控制台</td><td>莫里亚</td><td> 19360-3482 </td></tr></tbody></table>

corneal donor tissue preparation for endothelial keratoplasty

在过去的十多年中，角膜移植手术技术都发生了革命性的变化，1,2。公司自成立以来，一直是传统的全层角膜移植治疗恢复视力角膜疾病的限制。这种方法的一些缺点包括手术后散光的程度高，缺乏可预见的屈光结果，眼球表面的干扰。后弹力剥内皮角膜移植术（DSEK）移植后的角膜基质层，后弹力膜，和内皮细胞，发展已经极大地改变角膜内皮疾病的治疗。 DSEK通过一个小切口，其出血或驱逐的危险，这种技术避免了“开放天空”的手术，减少术后伤口裂开的发生率，减少不可预知的屈光结果，并可能会降低移植排斥反应率3-6。 ove_content“&gt;最初，眼角膜捐赠后为DSEK层状剥离，进行手动1产生的变量移植厚度和损坏的微妙的角膜组织处理过程中血管内皮组织。自动层状剥离（后弹力剥离自动化内皮角膜移植术，DSAEK）被开发，以解决这些自动清扫问题。利用机械角膜刀刀片，有助于创造DSAEK手术均匀且薄的组织移植与组织处理的角膜内皮细胞损失最小为LASIK术后角膜瓣制作技术。 眼库已提供多年移植手术的全层角膜。眼库在2006年开始开发供内皮角膜移植术的预切的角膜组织的方法。随着角膜外科医生的输入，眼银行已经开发了彻底的协议，安全和有效地准备后的层状组织f或的DSAEK手术。这可以进行术前眼库。研究表明，没有任何组织或患者的治疗效果为DSAEK手术中使用眼库预切组织与外科医生准备组织8,9质量方面的显着性差异。对于大多数角膜的外科医生，预切DSAEK角膜组织的可用性，节省了时间和金钱10，减少了在手术室进行捐助角膜剥离的压力。因为眼库的能力，及时地提供高品质的后板层角膜的一部分，，DSAEK已成为手术治疗角膜内皮病变的护理标准。 我们所描述的程序，是准备后在眼库的层状角膜移植在DSAEK手术的（ 图1）。

Watch this Scientific Journal Video about Corneal Donor Tissue Preparation for Endothelial Keratoplasty at JoVE.com

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

内皮角膜移植是治疗后角膜疾病的外科技术。机械角膜剥离，准备在更薄，更对称的移植组织的结果少内皮细胞丢失和改进的成果。夹层可以在眼库角膜移植手术前进行。

供体角膜内皮角膜移植术的组织准备

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

corneal-donor-tissue-preparation-for-endothelial-keratoplasty

Research

JoVE Journal

Medicine

27.4K 次观看.  University of Michigan. 内皮角膜移植是治疗后角膜疾病的外科技术。机械角膜剥离，准备在更薄，更对称的移植组织的结果少内皮细胞丢失和改进的成果。夹层可以在眼库角膜移植手术前进行。

视频: Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

单端口捐赠者肾切除术

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

科研

医学

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Corneal Donor Tissue Preparation for Descemet&#039;s Membrane Endothelial Keratoplasty

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

角膜供体组织准备后弹力层内皮角膜移植术

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

使用分裂的角质按钮的孔膜内皮器官培养模型

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

研究角膜伤口愈合的上皮擦伤模型

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

一种猪异位心脏移植方案，用于向心脏同种异体移植物提供治疗药物

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

长期培养用人心肌组织的制备

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

体内心脏移植研究内皮到间充质转化

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

作者聚焦：用于研究内皮-间充质转化的新生儿异位大鼠心脏移植模型  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

新生儿异位大鼠心脏移植模型在内皮间充质转化研究中的应用

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...