Name: growth of human and sheep corneal endothelial cell layers on biomaterial membranes
Uploaded: 2020-02-06T14:30:00
Description: Watch this Scientific Journal Video about Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes at JoVE.com

感谢诺埃米·加洛尼在编制图7期间给予的帮助。这项工作得到了澳大利亚国家卫生和医学研究委员会（项目赠款1099922）向卫生署提供的项目赠款以及昆士兰眼科基金会提供的补充资金的支持。

经捐赠者同意进行研究的人类角膜从昆士兰眼库获得，并经地铁南方医院和卫生服务部人类研究伦理委员会（HREC/07/QPAH/048）的道德认证使用。根据组织共享协议，在昆士兰大学赫斯顿医学研究设施从安乐死动物身上获得羊角膜。
1. 准备解剖工具
<ol><li>将两对 4 号制表匠钳浸泡在 70% 乙醇溶液中 5 分钟或高压灭菌，使其消毒。</li>
</ol>2. 培养培养基板和组织培...

<ol>
	<li>G&#252;ell, J. L., El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmology and Therapy. 3, 1-15 (2014).</li><li>Tan, D. T. H., Dart, J. K. G., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> The Lancet. 379 (9827), 1749-1761 (2012).</li><li>Soh, Y. Q., Peh, G. S. L., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+issues+for+human+corneal+endothelial+tissue+engineering.">Translational issues for human corneal endothelial tissue engineering.</a> Journal of Tissue Engineering and Regnerative Medicine. 11 (9), 2425-2442 (2017).</li><li>Senoo, T., Joyce, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Cycle+Kinetics+in+Corneal+Endothelium+from+Old+and+Young+Donors.">Cell Cycle Kinetics in Corneal Endothelium from Old and Young Donors.</a> Investigative Ophthalmology, Visual Science. 41 (3), 660-667 (2000).</li><li>Roy, O., Leclerc, V. B., Bourget, J. M., Th&#233;riault, M., Proulx, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+process+of+corneal+endothelial+morphological+change+in+vitro.">Understanding the process of corneal endothelial morphological change in vitro.</a> Investigative Ophthalmology, Visual Science. 56, 1228-1237 (2015).</li><li>Harkin, D. G., 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=Mounting+of+Biomaterials+for+Use+in+Ophthalmic+Cell+Therapies.">Mounting of Biomaterials for Use in Ophthalmic Cell Therapies.</a> Cell Transplantation. 26 (11), 1717-1732 (2017).</li><li>Trepat, X., Chen, Z., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration.">Cell migration.</a> Comprehensive Physiology. 2 (4), 2369-2392 (2012).</li><li>Walshe, J., Harkin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+explant+culture+provides+novel+insights+into+the+potential+location+and+phenotype+of+corneal+endothelial+progenitor+cells.">Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells.</a> Experimental Eye Research. 127, 9-13 (2014).</li><li>Al Abdulsalam, N. K., Barnett, N. L., Harkin, D. G., Walshe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+corneal+endothelial+cells+from+sheep.">Cultivation of corneal endothelial cells from sheep.</a> Experimental Eye Research. 173, 24-31 (2018).</li><li>Parekh, M., Ferrari, S., Sheridan, C., Kaye, S., Ahmad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+Review:+An+Update+on+the+Culture+of+Human+Corneal+Endothelial+Cells+for+Transplantation.">Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation.</a> Stem Cells Translational Medicine. 5 (2), 258-264 (2016).</li><li>Peh, G. S., Toh, K. P., Wu, F. Y., Tan, D. T., Mehta, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+human+corneal+endothelial+cells+isolated+from+paired+donor+corneas.">Cultivation of human corneal endothelial cells isolated from paired donor corneas.</a> PLoS One. 6 (12), 28310 (2011).</li></ol>

与建立和扩大人类角膜内皮细胞相关的一项重大技术挑战是防止EMT在培养物中发生。EMT可以通过细胞与细胞接触的丧失在角膜内皮细胞中触发，然而这些细胞的大多数细胞培养协议在分离和亚培养过程中涉及酶分离到单个细胞。在这里，我们提出了角膜内皮细胞的替代细胞培养方案，将细胞在隔离和亚培养阶段失去接触的风险降至最低。
我们建立角膜内皮?...

角膜是位于眼睛前面的透明组织。它由三个主要层组成：外表面的上皮层、中间的频闪层和称为角膜内皮的内层。角膜内皮是一个单层细胞，位于称为Descemet膜的基底膜上，通过调节从底层水性幽默进入频闪的液体量来保持角膜的透明度。频闪内过多的液体会导致角膜肿胀、不理想和视力丧失。因此，内皮内皮对维持视力至关重要。
角膜内皮可能由于多种原因（包括衰老、疾病和损伤）而功能失调，目前唯一的治疗是移植手术。在这次手术中，内皮和Descemet的膜从患者的角膜上去除，代之以从供体角膜获得的内皮和Descemet膜的移植物。许多内皮移植物也含有一层薄薄的基质组织，以帮助处理和附着到宿主角膜1。
在全球范围内，移植手术对角膜供体组织的需求大于眼库2所能提供的量。因此，人们一直在推动开发组织工程的角膜内皮移植，可用于缓解这一不足3。其依据是，目前，单个角膜的内皮只能转移到单个患者，但是，如果角膜内皮细胞首先在组织培养的生物材料支架上扩张和生长，它们可用于治疗多个患者。
在组织工程的角膜内皮移植成为外科医生的可行选择之前需要解决的主要挑战包括：（1） 建立扩展高质量角膜内皮细胞和生产成熟和功能性角膜内皮细胞层在体外，和（2）建立技术，在生物材料支架上生长细胞，以产生组织工程移植物，等于或优于目前使用的供体角膜衍生移植物。
角膜内皮细胞在体内具有非常低的增殖潜力，但可以刺激在体外分裂4。然而，他们有强烈的倾向，经历体外上皮到中皮过渡（EMT），这降低了他们的能力，形成一个成熟的，功能内皮层。角膜内皮细胞中EMT的已知诱因包括接触某些生长因子和细胞与细胞接触的丧失5。因此，在亚培养过程中酶分离的角膜内皮细胞培养物几乎是不可避免的，这种变化与EMT相关。在这里，我们提出了一种用于人类或绵羊角膜内皮细胞的细胞培养方法，该方法旨在最大限度地减少在隔离、扩张和亚培养阶段细胞与细胞接触的中断，以降低EMT的可能性。此外，我们演示了如何通过在定制安装装置中生物材料膜两侧生长培养细胞层来生成类似于供体角膜衍生内皮/Descemet 膜/体组织移植物的组织工程移植物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Attachment factor</td>
			<td>Gibco</td>
			<td>S006100</td>
			<td>A 1X sterile solution containing gelatin that is used to coat tissue culture surfaces. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Bovine pituitary extract</td>
			<td>Gibco</td>
			<td>13028014</td>
			<td>A single vial contains 25 mg. Freeze in aliquots.</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Merck</td>
			<td>C5670</td>
			<td>Dissolve in HBSS to make a 1 mM stock solution. Filter sterilise.</td>
		</tr>
		<tr>
			<td>Centrifuge tube, 50 ml</td>
			<td>Labtek</td>
			<td>650.550.050</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondroitin sulphate</td>
			<td>LKT Laboratories</td>
			<td>C2960</td>
			<td>This is bovine chondroitin sulphate. Dissolve in HBSS to make a 0.08 g/mL stock solution. Filter sterilise and freeze in aliquots.</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>Dissolve in DPBS to make a 2 mg/mL stock solution. Filter sterilise and freeze in aliquots.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Labtek</td>
			<td>EA043</td>
			<td>100% undenatured ethanol should be diluted to 70% in deionised water for sterilising instruments and surfaces.</td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>GE Healthcare Australia Pty Ltd</td>
			<td>SH30084.03</td>
			<td>This is a HyClone brand of foetal bovine serum.</td>
		</tr>
		<tr>
			<td>Coverglass No. 1, &#216; 13 mm</td>
			<td>Proscitech</td>
			<td>G401-13</td>
			<td>Place sterilised cover slips into 24-well plates for tissue culture.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>Hank&#39;s balanced salt solution, 1X, containing calcium chloride and magnesium chloride.</td>
		</tr>
		<tr>
			<td>L-ascorbic acid 2-phosphate</td>
			<td>Merck</td>
			<td>A8960</td>
			<td>Dissolve in HBSS to make a 150 mM stock solution. Filter sterilise.</td>
		</tr>
		<tr>
			<td>Micro-Boyden chamber</td>
			<td>CNC Components Pty. Ltd.</td>
			<td>Upper ring: QUT-0002-0006, Base ring: QUT-0002-0007</td>
			<td>Both components are made from polytetrafluoroethelyne (PTFE).</td>
		</tr>
		<tr>
			<td>O-ring for micro-Boyden chamber</td>
			<td>Ludowici Sealing Solutions</td>
			<td>RSB012</td>
			<td>Composed of silicon rubber.</td>
		</tr>
		<tr>
			<td>Opti-MEM 1 (1X) + GlutaMAX-1</td>
			<td>Gibco</td>
			<td>51985-034</td>
			<td>A reduced serum medium containing glutamine.</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Dulbecco&#39;s phosphate buffered saline, 1X, without calcium chloride and magnesium chloride.</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>A 100X antibiotic solution containing 10,000 Units/mL penicillin and 10,000 &#181;g/mL streptomycin.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82.14473.001</td>
			<td>Sterile Petri dish, 92 X 16 mm, for tissue dissections.</td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 well</td>
			<td>Corning Incorporated</td>
			<td>Costar 3524</td>
			<td>A plate containing 24 wells, each with a surface area of 2 cm2.</td>
		</tr>
		<tr>
			<td>Tissue culture plate, 6 well</td>
			<td>Corning Incorporated</td>
			<td>Costar 3516</td>
			<td>A plate containing 6 wells, each with a surface area of 9 cm2.</td>
		</tr>
		<tr>
			<td>TrypLE Select</td>
			<td>Gibco</td>
			<td>12563-011</td>
			<td>A 1X enzyme solution for dissociating cells.</td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Gibco</td>
			<td>15040-066</td>
			<td>A 1X EDTA solution for dissociating cells.</td>
		</tr>
		<tr>
			<td>Watchmaker forceps</td>
			<td>Labtek</td>
			<td>BWMF4</td>
			<td>Number 4 watchmaker forceps work well for removing strips of endothelium/Descemet&#39;s membrane from corneas.</td>
		</tr>
	</tbody>
</table>

growth of human and sheep corneal endothelial cell layers on biomaterial membranes

角膜内皮细胞培养体在细胞与细胞失去接触后有发生上皮到中皮过渡（EMT）的倾向。EMT对细胞是有害的，因为它降低了细胞形成成熟和功能层的能力。在这里，我们提出了一种建立和亚培养人类和绵羊角膜内皮细胞培养的方法，以尽量减少细胞与细胞接触的损失。角膜内皮/Descemet膜的外部从供体角膜中取走，在允许细胞集体迁移到培养表面的条件下放入组织培养中。一旦文化建立，外植物被转移到新鲜的板块，以启动新的文化。去巴塞II用于轻轻地将细胞团从组织培养板上提起，用于亚培养。使用该协议建立的角膜内皮细胞培养物适用于转移到生物材料膜，以产生组织工程细胞层，用于动物试验中的移植。介绍了一种在组织培养过程中支持生物材料膜的定制装置，并举例说，由一层角膜内皮细胞和胶原蛋白I型膜两侧的角膜基质细胞组成的组织工程移植物。

图1和图2总结了从人或羊角膜中分离和扩大角膜内皮细胞的方法。大多数来自1至2岁绵羊角膜或30岁以下人类捐赠者的外植植物将在一周内附着在附着因子组织培养板上，然而，发现多达三分之一的外科在这段时间内无法附着，这种情况并不罕见。这些"浮动"的外植可以从培养物中去除。超过30年的人类捐赠者的外植不太可能附着在板上，也不太可能产生...

Watch this Scientific Journal Video about Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes at JoVE.com

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

该协议描述了从人类或绵羊组织外植植物中建立和生长角膜内皮细胞培养的关键步骤。提出了一种在膜状生物材料上对角膜内皮细胞进行亚培养的方法。

生物材料膜上人与羊角膜内皮细胞层的生长

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

此细胞培养协议旨在防止角膜内皮细胞在隔离、扩张和亚培养期间经历上皮到中层过渡。细胞培养物由角膜组织外植物建立，可重复使用，以确保从一个捐赠者持续供应原细胞。首先，使用制造商的说明，用附件因子为六孔组织培养板的孔涂上涂层。然后向每个编码良好的培养介质添加一毫升的培养介质。将角膜内皮面向上放入解剖显微镜的舞台上的无菌培养皿中。调整照明，使角膜表面照明良好，具有足够的对比度，以突出内皮层，并调整变焦，使边缘和一些中央角膜内皮在视图中。使用灭菌制表器钳子轻轻地将 Descemet 的膜从底层频闪中提起并撕裂。将膜条放入培养板的准备孔之一，确保立即更换板盖，以降低污染风险。首先从内皮的极端边缘取出 Descemet 膜条，然后从中心区域取出，将一个角膜的所有条状物放入一个孔中。完成后，在加湿细胞培养孵化器中孵育出的植物设定为37摄氏度和5%的二氧化碳。将微型博伊登室的上室组装，将红色 O 环放入其中心。使用直径为 18 毫米的树外，从 PTFE 切割板上的生物材料片中打出圆盘。然后，将磁盘放在微型博伊登室的上室的红色 O 环上。将下腔拧到上腔室上，将生物材料的盘固定在两者之间。将组装好的微型博伊登腔室浸泡在70%乙醇中一小时，进行消毒。浸泡后，将腔室完全浸入无菌的HSSS中10分钟，取出乙醇。然后用培养介质清洗腔室10分钟。并转移到六井板井内的培养介质，确保上室朝向。调整介质的水平，使介质接触生物材料膜的下表面，但不会流入上室。将100微升细胞悬浮液移入膜中，放入微型博伊登腔室，并在组织培养培养箱中孵育4小时，然后加入介质完全淹没腔室。大多数从一至两岁绵羊的角膜或小于30岁的人类捐赠者身上衍生出的植物，附着在一周内附着在附着因子编码的组织培养板上。六周后，许多小而密闭的细胞出现在外植体旁边。角膜内皮细胞培养中的小型、紧密包装的细胞对三重消化具有抵抗力，而较大的成纤维细胞则对消化更敏感。这种差异被利用来选择性地从培养物中去除大细胞，然后再将较小的细胞转移到新的细胞中。对角膜内皮细胞培养物进行了免疫粉化分析，以找到ZO-1，一种紧密结蛋白，和N-卡德林，一种粘结蛋白。同样的抗体用于绵羊和人类细胞，在这两种细胞的血浆膜中检测到这两种蛋白质。定制的微型博伊登腔室允许膜的两侧同时用作细胞培养表面。组织工程细胞构造的横截面视图显示一个表面上的角膜内皮细胞的单层，另一个表面的角膜状细胞的多层培养。尝试此协议时，当您将 Descemet 的膜从角膜上剥去时，避免用钳子刮除内皮非常重要。按照此过程，可以通过测量膜上的角膜内皮细胞层的完整性来测量其电阻。

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In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

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Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

铁磁金属裸支架对血管内皮细胞捕获和保留

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

白细胞 - 富血小板纤维蛋白，一种新型生物材料的表征

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

人卵巢组织与工程化内皮细胞联合移植: 一种以细胞为基础的快速灌注与直接分泌的融合策略

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

巨噬细胞报告器细胞分析，以检查聚合物表面的吸附蛋白层上的收费类似受体介导NF-kB/AP-1信号

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...