1，建立无菌区的<ol><li>唐合适的服装，包括帽子和口罩。戴防护眼镜，在处理室时。 </li><li>准备处理区域。当使用一个加工室，维持风扇的连续操作，以创建在室内的正压。 </li><li>收集所有用品。检查到期日期，包装完整性，灭菌指标。 </li><li>洗手，不要非无菌手套。 </li><li>打开无菌包，以建立一个无菌区。当使用一个加工室，放在桌上的包和无菌条件下打开包裹。不利用外部1.5英寸的卷体的无菌区的一部分，因为这部分被认为是未消毒。 </li><li>使用无菌技术，开放的拖放无菌物品到无菌区，除非预先做好的包已经组成部分。 </li><li>移动显微镜，使其上面的无菌区域，打开显微镜的光源，并调整目镜是必要的。 </li><li> OBTAIN角膜组织被剥离（在保存介质），并置于旁边的制备无菌处理区域。松开观察室的盖子，让它停留在观察室的基础，没有搞线程的顶部。丢弃非无菌手套。 </li><li>打开一对无菌手套和无菌袍毛巾。 </li><li>进行手术擦洗。穿上无菌衣，再用无菌手套。 </li><li>打开仪器箱，让所有用品都可以访问。打开水不渗透的悬垂性，以创建工作区。 </li><li>附针5毫升注射器。使用止血钳，通过从斜角放置一个弯曲1毫米制备地下​​30针，并从基体的另一个弯曲1毫米（以形成一"S"形状）。 </li><li>通过将柱塞插入注射器和手指拧紧柱塞组装玻璃注射器的台盼蓝染色。连接套管。 </li><li>连接套管的平衡盐溶液（BSS）的瓶子。 </li><li>从真空块分开的环钻打孔并预留。视显微镜的视野内居中真空块。 </li><li>通过调整显微镜的设置带来的真空块成为关注焦点。 </li></ol> 2，程序供体组织准备<ol><li>观察室使用方形铝箔作为阻隔的掀盖。用另一只手使用镊子，轻轻抓住了角膜巩膜轮辋去除角膜。更换观察室的盖子。 </li><li>完全踩下吸入注射器中，然后将角膜上皮侧朝下的真空块上，并确保它的中心。 （ 图1） </li><li>在使用导柱真空块轻轻下休息环。慢慢松开吸入注射器的活塞从事真空。拆下座椅环，备用。 （ 图2）。 </li><li>执行部分环钻术，以高分通过后弹力膜。 </li><li>调整显微镜变焦和聚焦，使该组织及可视化。 </li><li&gt;倾斜真空块，并用无菌棉签矛放置在角膜缘以除去多余的介质。 （ 图3） </li><li>将足够的染色在角膜缘染色的进球后弹力膜的边缘。允许污渍保留在血管内皮细胞为60-90秒。 （ 图4） </li><li>移除污点: <ol><li>倾斜真空块倒污垢角膜和上纱布垫。 </li><li>用手术长矛从角膜缘区域去除多余的污渍，注意不要接触内皮细胞。 （ 图5） </li></ol></li><li>用几滴BSS的冲洗染色液角膜，无菌棉签长矛，去除多余的BSS。 （ 图6） </li><li>放置在巩膜缘1-2滴BSS以防止组织干燥。根据需要重复整个过程。 </li><li>执行后弹力膜部分剥离: <ol><li>开始当管路连接s到真空块，使用开放镊子，将一个尖的得分标记的边缘，并轻轻分开后弹力膜和内皮远离基质。 （ 图7）。 </li><li>分开后弹力膜和内皮细胞不超过1毫米的得分边缘和继续剥离360°左右。 （ 图8）。 </li><li>注意任何微小的泪水位置放置点与皮肤标记真空块。 注意:如果该环钻没有通过后弹力膜完全渗透，使用所制备的地下30针的斜面边缘，完成通过后弹力膜在有需要时，随后染色的第二应用在需要的分数。如果后弹力膜的粘附性是阻碍局部解剖，根据需要取出trephinated禁区外的内皮细胞。 </li></ol></li><li>使用镊子去除重叠的得分标记任何内皮标记。 </li> &lt;LI&gt;考虑了先前标记的微撕裂的位置和旋转真空块，使最大的是位于6点钟位置。这将成为护翼的铰链。 </li><li>使用镊子，抓住后弹力膜在12点。轻轻剥离朝铰链分开后弹力膜的基质。通过停止从得分标记分离2毫米创建的铰链。 注:瓣的周缘开始分离过程中进行滚动。 （ 图9）。 </li><li>轻轻地将瓣放回原处的基质。用BSS和拭子长矛unscroll的内皮细胞，并将其返回到其原始位置。 （ 图10A-C） </li><li>从后弹力膜和基质之间用无菌棉签长矛删除这样的襟翼保持在适当的位置残留的液体。 （ 图11） </li><li>用消毒棉签长矛来吸收多余的水分从靠近转轴处的巩膜。使用皮肤标记来绘制Ñ​​慈姑上巩膜缘指向铰合部的中心，用无菌拭子矛除去任何过量的墨水。 （ 图12） </li><li>通过按压连接到真空块状注射器的活塞释放真空。而真空被切断，使用镊子从真空块除去角膜。 </li><li>观察室使用方形铝箔作为阻隔的掀盖。放置在角膜的观察室，内皮面朝上，并取代观察室的盖子。 </li><li>重复上面的操作步骤与队友角膜（如适用）。用纱布处理所述第二组织前清洁真空块。 </li><li>拧紧的观看室盖（次），并从处理区域除去组织。 </li><li>通过根据生物危害废物管理计划处置项目分解无菌区。脱去污染的手套和礼服。洗手，不要非无菌手套。 </li><li>进行后切裂隙灯评价和角膜的镜面反射镜（次）并记录结果。 </li></ol>

<ol>
	<li>Fernandez, M., Afshari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+From+DLEK+to+DMEK.">Endothelial Keratoplasty: From DLEK to DMEK.</a> Middle East African Journal of Ophthalmology. 17 (1), 5-8 (2010).</li><li>Kruse, F., 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=A+Stepwise+Approach+to+Donor+Preparation+and+Insertion+Increases+Safety+and+Outcome+of+Descemet+Membrane+Endothelial+Keratoplasty.">A Stepwise Approach to Donor Preparation and Insertion Increases Safety and Outcome of Descemet Membrane Endothelial Keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Dapena, I., Moutsouris, K., Droutsas, K., Ham, L., van Dijk, K., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+'No-Touch'+Technique+for+Descemet+Membrane+Endothelial+Keratoplasty.">Standardized 'No-Touch' Technique for Descemet Membrane Endothelial Keratoplasty.</a> Archives of Ophthalmology. 129, 88-94 (2011).</li><li>Tourtas, T., Laaser, K., Bachmann, B., Cursiefen, C., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+Endothelial+Keratoplasty+versus+Descemet+Stripping+Automated+Endothelial+Keratoplasty.">Descemet Membrane Endothelial Keratoplasty versus Descemet Stripping Automated Endothelial Keratoplasty.</a> American Journal of Ophthalmology. 153 (6), 1082-1090 (2012).</li><li>Guerra, F., Anshu, A., Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+fellow+eyes+comparison+of+Descemet+stripping+automated+endothelial+keratoplasty+and+Descemet+membrane+endothelial+keratoplasty.">Endothelial Keratoplasty: fellow eyes comparison of Descemet stripping automated endothelial keratoplasty and Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (12), 1382-1386 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2012 Eye Banking Statistical Report: Surgical Use and Indications for Corneal Transplant Statistical Report Analysis. , (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+stripping+endothelial+keratoplasty.">Descemet's stripping endothelial keratoplasty.</a> Current Opinion Ophthalmology. 18 (4), 290-294 (2007).</li><li>Woodward, M., Titus, M., Mavin, K., Shtein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Donor+Tissue+Preparation+for+Endothelial+Keratoplasty.">Corneal Donor Tissue Preparation for Endothelial Keratoplasty.</a> Journal of Visualized Experiments. (64), e3847 (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+membrane+endothelial+keratoplasty+surgery:+update+on+the+evidence+and+hurdles+to+acceptance.">Descemet's membrane endothelial keratoplasty surgery: update on the evidence and hurdles to acceptance.</a> Current Opinion Ophthalmology. 24 (4), 329-335 (2013).</li><li>Venzano, D., Pagani, P., Randazzo, N., Cabiddu, F., Traverrso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+air-bubble+separation+in+donor+cornea.">Descemet Membrane air-bubble separation in donor cornea.</a> Journal of Cataract and Refractive Surgery. 36 (12), 2022-2027 (2010).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DSEK: What you need to know about endothelial keratoplasty. , (2009).</li><li>Studeny, P., Sivekova, D., Lieheova, K., Vokrojova, M., Kuchynka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+technique+of+lamellar+keratoplasty+(DMEK-S).">Hybrid technique of lamellar keratoplasty (DMEK-S).</a> Journal of Ophthalmology. 2013, 2013.254383 (2013).</li><li>Muraine, M., Gueudry, J., He, Z., Piselli, S., Lefevre, S., Toubeau, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+technique+for+the+preparation+of+corneal+grafts+for+Descemet+membrane+endothelial+keratoplasty.">Novel technique for the preparation of corneal grafts for Descemet membrane endothelial keratoplasty.</a> American Journal of Ophthalmology. 156 (5), 851-859 (2013).</li><li>Lie, J. T., Birbal, R., Ham, L., van der Wees, J., Melles, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+tissue+preparation+for+Descemet+membrane+endothelial+keratoplasty.">Donor tissue preparation for Descemet membrane endothelial keratoplasty.</a> Journal of Cataract Refractive Surgery. 34 (9), 1578-1583 (2008).</li><li>Kruse, F. E., Laaser, K., Cursiefen, C., Heindl, L. M., Schlotzer-Schrehardt, U., Riss, S., Bachmann, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+approach+to+donor+preparation+and+insertion+increases+safety+and+outcome+of+Descemet+membrane+endothelial+keratoplasty.">A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Rudolph, M., Kaaser, L., Bachmann, B., Cursiefen, C., Epstein, D., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+higher-order+aberrations+after+Descemet&#8217;s+membrane+endothelial+keratoplasty.">Corneal higher-order aberrations after Descemet&#8217;s membrane endothelial keratoplasty.</a> Ophthalmology. 119 (3), 528-535 (2012).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+endothelial+keratoplasty.">Evolution of endothelial keratoplasty.</a> Cornea. 32, 28-32 (2013).</li><li>Dapena, I., Dapena, L., Dirisamer, M., Ham, L., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+and+endothelial+cell+density+following+Descemet+membrane+endothelial+keratoplasty+(DMEK).">Visual acuity and endothelial cell density following Descemet membrane endothelial keratoplasty (DMEK).</a> Arch Soc Esp Oftalmol. 86 (12), 395-401 (2011).</li></ol>

哈桑Tausif和Navasuja Chandrasekaran博士什么都没有透露。博士。勉，Shtein和伍德沃德是医疗董事密歇根州眼库。 Mavin先生，提多先生和Johnson女士是中西部眼库的员工。

角膜内皮细胞层起着通过脱水维持角膜透明度的关键作用。内皮细胞的疾病都可以导致显著视力障碍。 DMEK旨在与供体移植物，包括后弹力层和内皮细胞层取代病变血管内皮细胞。供体组织编制了一个完整的DM和足够的内皮细胞计数是移植组织的生存至关重要。糖尿病供体组织筹备过程中撕裂可导致组织损失和捐助组织的浪费。 DMEK的广泛应用已经在准备移植的组织和掌握复杂的手术技术所面临的?...

角膜内皮移植术（EK）已经迅速取代于2012年6穿透性角膜移植术（PKP）作为首选治疗内皮疾病占美国移植手术的50％以上的步骤。PKP是由长期的视觉康复高度散光的限制和屈光不稳定，全球诚信和缝线相关并发症的损失。初代EK程序，包括深板层内皮角膜（DLEK）参与角膜基质偏厚供体组织移植的人工清扫。这些程序成功地移植供体血管内皮组织，同时保持一个完整的角膜表面，但在技术上具有挑战性，由于不规则角膜板层接口，1导致有限的最佳矫正视力。选择目前EK程序后弹力层剥除内皮角膜移植术（DSEK），提供角膜CLAR性与更快的恢复视力，减少术后屈光度，散光，并降低外伤伤口裂开的风险，同时保持角膜表面7。此过程涉及去除病变后弹力膜和内皮细胞和置换部分的厚度供体角膜组织，包括一个新的内皮细胞层。供体角膜的解剖与角膜板层刀来准备后组织被移植8。由眼银行的供体组织的制备一直在快速增长，可以说，这个程序的成功。 2005年至2012年的近16倍的DSEK程序在美国对应目前正在编写的眼睛6银行组织的近一半。 虽然DSEK可以提高视力，以20/40以上，多数患者没有达到正常视力，由于间质混浊的界面和增强的光从另外捐助基质组织7的散落一地。该EK手术技术已经发展到后弹力膜内皮角膜移植术（DMEK）涉及的只给予体后弹力膜（DM）和血管内皮细胞的移植。本程序有谁达到正常视力，视力恢复快，而血管内皮抑制9的风险较低的患者比例较高。然而，准备供体组织是更具挑战性的是由于移植物更薄的尺寸。

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:268px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">VisionBlue&#160;</td>
			<td style="width:268px;">D.O.R.C., Zuiland, The Netherlands</td>
			<td style="width:119px;">NA</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Barron vacuum punch</td>
			<td style="width:268px;">Katena, Denville, New Jersey</td>
			<td style="width:119px;">K20-2112</td>
			<td></td>
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corneal donor tissue preparation for descemet's membrane endothelial keratoplasty

后弹力膜内皮角膜移植术（DMEK）是角膜移植，其中只有一个单细胞层，角膜内皮细胞，连同其基底膜（Descemet膜）被引入到接收方的后基质3的形式。与角膜后弹力层剥除自动内皮角膜移植术（DSAEK），其中更多的捐助基质的介绍，不自然基质对基质的界面创建。其结果，角膜的天然解剖学保留尽可能允许改善恢复时间和视力4。角膜内皮移植术（EK）是首选的治疗内皮功能障碍的过程。 EK的优点包括快速恢复视力，保护眼部的完整性和最小的屈光变化，由于采用了小型，外围切口1。 DSAEK利用制备偏厚的基质和内皮细胞供体组织。快速SUCCESS和利用这个程序可以归因于眼睛银行可用性制备预切割的组织。眼库准备供体组织的好处包括:消除需要专门的设备备份供体组织的情况下，组织穿孔制备过程中的手术室和可用性。此外，高体积制备由眼库的技术人员供体组织可以提供供体组织的改进的质量。 DSAEK可能限制最佳矫正视力，由于创作的供体和受体角膜之间的间质界面。消除这个接口仅为捐助后弹力膜和内皮DMEK移植可以改善视觉效果和EK 5后减少并发症的发生。类似DSAEK，长期成功和验收DMEK的是依赖于易于预切割的可用性，眼库制备供体组织。在这里，我们提出了一个阶梯式的供体组织编写这可能会降低一些巴里ERS眼银行面临提供DMEK移植。

从基质适当分离，DM和内皮正在准备预切组织的DMEK必不可少的。五十捐献角膜为65±6岁（范围55-75），死亡2,616±321个/ mm 2至5.9±2.3天（范围1-10）和内皮细胞密度的处理时间（ECD）（平均年龄范围2,049-3,247）被用于由两个眼库的技术人员来评估手术制备技术的可行性和可重复性。成功率为72％，前25例，上升至80％，下半年76％的成功率。点二列相关（RPB）来确定，术前组织因子的相关性。捐?...

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

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

角膜内皮移植术是一种外科技术不断发展的进步导致的移植物薄，每个继承1移植。在这里，我们提出了一个技术控制的手动组织剥离，使血管内皮和后弹力捐助角巩膜按键膜移植2的安全和可重复的分离。

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, ...

corneal-donor-tissue-preparation-for-descemet-s-membrane-endothelial

Research

JoVE Journal

Medicine

15.2K 次观看.  University of Michigan. 角膜内皮移植术是一种外科技术不断发展的进步导致的移植物薄，每个继承1移植。在这里，我们提出了一个技术控制的手动组织剥离，使血管内皮和后弹力捐助角巩膜按键膜移植2的安全和可重复的分离。 

视频: Corneal Donor Tissue Preparation for Descemet's Membrane 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 Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

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 ...

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 ...

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 ...

Rodent Heart and Brain Tissue Preparation for Digital Macro Photography after Ischemia-reperfusion

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue preparation and subsequent image capture are steps performed immediately after the ischemia-reperfusion (IR) experiment and must be performed in a timely manner and with appropriate care. For the evaluation of IR-induced damage in the heart and brain, this paper describes 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)-based staining followed by macro photography. Scientific macro photography requires controlled lighting and an appropriate imaging setup. The standardized methodology ensures high-quality, detailed digital images even if a combination of an inexpensive up-to-date digital camera and macro lens is used. Proper techniques and potential mistakes in sample preparation and image acquisition are discussed, and examples of the influence of correct and incorrect setups on image quality are provided. Specific tips are provided on how to avoid common mistakes, such as overstaining, improper sample storage, and suboptimal lighting conditions. This paper shows the appropriate methodology for rat heart and brain tissue slicing and staining and provides guidelines for establishing lighting and camera setups and photography techniques for high-resolution image acquisition.

Presented here is a protocol for the standardized methodology of rodent tissue preparation after the ischemia-reperfusion experiment and guidelines for establishing lighting and camera setups for high-resolution image acquisition. This method is applicable to all experimental small-animal organ photography.

啮齿动物心脏和脑组织准备缺血后数码微距摄影

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue ...

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 ...