Name: a porcine corneal endothelial organ culture model using split corneal buttons
Uploaded: 2019-10-06T14:00:00
Description: Watch this Scientific Journal Video about A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons at JoVE.com

所提供的研究模型的建立得到了德国联邦教育和研究部的KMU-创新（FKZ：13GW0037F）的支持。

本协议遵循我们机构的道德准则。根据我们机构道德审查委员会的章程，在实验之前无需获得道德批准，因为所有猪角膜都是从当地屠宰场获得的。
1. 器官文化
<ol><li>准备猪眼。<ol>
<li>从当地的屠宰场，获得猪眼，在验尸后，但在热处理之前被摘除。将眼睛转移到实验室，并在几个小时内处理它们。在运输过程中，在加工前将眼睛保持在室温（约21°C） 下。</li>
		<li>使用眼...

<ol>
	<li>Gain, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Survey+of+Corneal+Transplantation+and+Eye+Banking.">Global Survey of Corneal Transplantation and Eye Banking.</a> JAMA Ophthalmology. 134 (2), 167-173 (2016).</li><li>Roy, O., 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=Understanding+the+process+of+corneal+endothelial+morphological+change+in+vitro.">Understanding the process of corneal endothelial morphological change in vitro.</a> Investigative Ophthalmology &amp; Visual Science. 56 (2), 1228-1237 (2015).</li><li>Meltendorf, C., Ohrloff, C., Rieck, P., Schroeter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+density+in+porcine+corneas+after+exposure+to+hypotonic+solutions.">Endothelial cell density in porcine corneas after exposure to hypotonic solutions.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 245 (1), 143-147 (2007).</li><li>Schroeter, J., Meltendorf, C., Ohrloff, C., Rieck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+temporary+hypothermia+on+corneal+endothelial+cell+density+during+organ+culture+preservation.">Influence of temporary hypothermia on corneal endothelial cell density during organ culture preservation.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 246 (3), 369-372 (2008).</li><li>Schroeter, J., Ruggeri, A., Thieme, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+temporary+hyperthermia+on+corneal+endothelial+cell+survival+during+organ+culture+preservation.">Impact of temporary hyperthermia on corneal endothelial cell survival during organ culture preservation.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 253 (5), 753-758 (2015).</li><li>Kunzmann, B. C., 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=Establishment+Of+A+Porcine+Corneal+Endothelial+Organ+Culture+Model+For+Research+Purposes.">Establishment Of A Porcine Corneal Endothelial Organ Culture Model For Research Purposes.</a> Cell and Tissue Banking. 19 (3), 269-276 (2018).</li><li>Redbrake, C., 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+histochemical+study+of+the+distribution+of+dextran+500+in+human+corneas+during+organ+culture.">A histochemical study of the distribution of dextran 500 in human corneas during organ culture.</a> Current Eye Research. 16 (5), 405-411 (1997).</li><li>Zhao, M., 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=Poloxamines+for+Deswelling+of+Organ-Cultured+Corneas.">Poloxamines for Deswelling of Organ-Cultured Corneas.</a> Ophthalmic Research. 48 (2), 124-133 (2012).</li><li>Filev, 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=Semi-quantitative+assessments+of+dextran+toxicity+on+corneal+endothelium:+conceptual+design+of+a+predictive+algorithm.">Semi-quantitative assessments of dextran toxicity on corneal endothelium: conceptual design of a predictive algorithm.</a> Cell and Tissue Banking. 18 (1), 91-98 (2017).</li><li>Pels, E., Schuchard, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-culture+preservation+of+human+corneas.">Organ-culture preservation of human corneas.</a> Documenta Ophthalmologica. 56 (1-2), 147-153 (1983).</li><li>Borderie, V. 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J., 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=Thirty+years+of+cornea+cultivation:+long-term+experience+in+a+single+eye+bank.">Thirty years of cornea cultivation: long-term experience in a single eye bank.</a> Acta Opthalmologica. 91 (6), 571-578 (2013).</li><li>Schroeter, J., 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=Arbeitsrichtlinien+-+Gute+Fachliche+Praxis+f&#252;r+Hornhautbanken+[Procedural+guidelines.+Good+tissue+practice+for+cornea+banks].">Arbeitsrichtlinien - Gute Fachliche Praxis f&#252;r Hornhautbanken [Procedural guidelines. Good tissue practice for cornea banks].</a> Ophthalmologe. 106 (3), 265-276 (2009).</li><li>Dohlman, C. H., Hedbys, B. O., Mishima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+swelling+pressure+of+the+corneal+stroma.">The swelling pressure of the corneal stroma.</a> Investigative Ophthalmology &amp; Visual Science. 1, 158-162 (1962).</li><li>Xuan, M., 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=Proteins+of+the+corneal+stroma:+importance+in+visual+function.">Proteins of the corneal stroma: importance in visual function.</a> Cell and Tissue Research. 364 (1), 9-16 (2016).</li><li>Sperling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Corneal+Endothelium+in+Organ+Culture+-+The+Influence+of+Temperature+and+Medium+of+Incubation.">Human Corneal Endothelium in Organ Culture - The Influence of Temperature and Medium of Incubation.</a> Acta Opthalmologica. 57 (2), 269-276 (1979).</li><li>Schroeter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Evaluation+in+the+Cornea+Bank.">Endothelial Evaluation in the Cornea Bank.</a> Developments in Ophthalmology. 43, 47-62 (2009).</li><li>Pels, E., Schuchard, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+High+Molecular+Weight+dextran+on+the+Presevation+of+Human+Corneas.">The Effects of High Molecular Weight dextran on the Presevation of Human Corneas.</a> Cornea. 3 (3), 219-227 (1985).</li><li>van der Want, H. J. L., Pels, E., Schuchard, Y., Olesen, B., Sperling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Microscopy+of+Cultured+Human+Corneas+Osmotic+Hydration+and+the+Use+of+dextran+Fraction+(dextran+T+500)+in+Organ+Culture.">Electron Microscopy of Cultured Human Corneas Osmotic Hydration and the Use of dextran Fraction (dextran T 500) in Organ Culture.</a> Archives of Ophthalmology. 101 (12), 1920-1926 (1983).</li><li>Thuret, G., Manissolle, C., Campos-Guyotat, L., Guyotat, D., Gain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+compound-free+medium+and+poloxamer+for+human+corneal+organ+culture+and+Deswelling.">Animal compound-free medium and poloxamer for human corneal organ culture and Deswelling.</a> Investigative Ophthalmology &amp; Visual Science. 46 (3), 816-822 (2005).</li><li>Wenzel, D. A., Kunzmann, B. C., Spitzer, M. S., Schultheiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+endothelial+cells+does+not+change+the+result+of+cell+density.">Staining of endothelial cells does not change the result of cell density.</a> Cell and Tissue Banking. 20 (2), 327-328 (2019).</li><li>Wenzel, D. A., Kunzmann, B. C., Hellwinkel, O., Druchkiv, V., Spitzer, M. S., Schultheiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+perfluorobutylpentane+(F4H5)+on+corneal+endothelial+cells.">Effects of perfluorobutylpentane (F4H5) on corneal endothelial cells.</a> Current Eye Research. , (2019).</li><li>Olsson, I. A. S., Franco, N. H., Weary, D. M., Sand&#248;e, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3Rs+principle+-+mind+the+ethical+gap!.">The 3Rs principle - mind the ethical gap!.</a> ALTEX Proceedings, 1/12, Proceedings of WC8. , 333-336 (2012).</li><li>Sanchez, I., Martin, R., Ussa, F., Fernandez-Bueno, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parameters+of+the+porcine+eyeball.">The parameters of the porcine eyeball.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 249 (4), 475-482 (2011).</li><li>Kim, M. K., Hara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+corneal+xenotransplantation.">Current status of corneal xenotransplantation.</a> International Journal of Surgery. 23 (Pt B), 255-260 (2015).</li><li>Fujita, M., 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=Comparison+of+Proliferative+Capacity+of+Genetically-Engineered+Pig+and+Human.">Comparison of Proliferative Capacity of Genetically-Engineered Pig and Human.</a> Ophthalmic Research. 49 (3), 127-138 (2013).</li></ol>

该协议提供了一种制备猪分裂角膜纽扣的方法，它代表了一种标准化和低成本的外体角膜内皮器官培养模型，用于研究目的6。猪分裂角膜纽扣显示内皮细胞密度下降，与在眼库培养的人类供体角膜中观察到的内皮细胞损失相当，在两周内6，10，11， 12.
优于非分?...

角膜移植手术是全世界最常见的移植之一。由于人类供体角膜严重短缺，人类角膜内皮细胞的实验研究难以进行。然而，引进灌溉溶液和眼睛内使用的其他物质，眼科粘弹性装置，以及手术器械和技术（例如，乳化仪器和技术，超声波能量）需要临床使用前，对角膜内皮药的影响进行有效和广泛的调查。
除了人类供体角膜之外，几乎没有其他选择可供研究。动物研究模型是非常有价值的，但与此同时，非常耗费资源，并越来越质疑伦理。体外细胞培养的一个主要缺点是它们对人眼的翻译有限。从细胞培养得到的结果可能与体内条件不协调，因为细胞可能经历内皮性等位体过渡（EMT），导致细胞极性丧失和细胞形状和基因变化引起的成纤维细胞样形态表达式2.
而以前的前体模型报告培育期只有120小时，一种新的制备技术，以建立猪角膜内皮器官培养模型，通过培育新鲜猪角膜至少15天最近推出3 ，4，5，6.如果在种植前从角膜上皮和频闪部分（总计约300 μm）去除角膜，则角质的肿胀在分裂的角膜按钮中减少，从而减少内皮细胞损失，并维持良好的内皮细胞层后长达15天，而非分裂的角膜按钮显示显着的内皮细胞损失，由于不均匀的基质肿胀和德塞梅特的褶皱的形成。眼库通常使用渗透消肿剂，如dextran，以减少在移植前角膜肿胀。然而，这些制剂被证明诱导增加的内皮细胞损失7，8，9。
本文旨在以详细的分步协议来可视化这种标准化的体外研究模型，以便未来的研究者能够使用分割角膜按钮对角膜内皮进行研究。该模型代表一种简单的方法来测试眼睛中使用的物质和技术，如眼粘弹性装置、灌溉溶液和超声波能量，或角膜内皮感兴趣的其他程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Subject</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pig eyes</td>
			<td>local abbatoir</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Substances</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red S</td>
			<td>Sigma-Aldrich, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Medium 1, #F9016</td>
			<td>Biochrom GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (1x)</td>
			<td>Gibco, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Biochrom GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl) solution</td>
			<td>own production</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypotonic balanced salt solution</td>
			<td>own production</td>
			<td></td>
			<td>per 1 L of H2O: NaCl 4.9 g; KCl 0.75 g; CaCl x H2O 0.49 g; MgCl2 x H2O 0.3 g; Sodium Acetate x 3 H2O 3.9 g; Sodium Citrate x 2 H2O 1.7 g</td>
		</tr>
		<tr>
			<td>Povidon iodine 7.5%, Braunol</td>
			<td>B. Braun Melsungen AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl) 0.9%</td>
			<td>B. Braun Melsungen AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH) solution</td>
			<td>own production</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>Sigma-Aldrich, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials &#38; Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accu-jet pro</td>
			<td>Brand GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker Glass 50 mL</td>
			<td>Schott AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt cannula incl. Filter (5 &#181;m) 18G</td>
			<td>Becton Dickinson, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plate (12 well)</td>
			<td>Corning Inc., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri forceps</td>
			<td>Geuder AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corneal scissors</td>
			<td>Geuder AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf pipette</td>
			<td>Eppendorf AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Bulb Holder</td>
			<td>L. Klein, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eye scissors</td>
			<td>Geuder AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Folded Filter &#248; 185 mm</td>
			<td>Whatman, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hockey knife</td>
			<td>Geuder AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Glass Bottle with cap 100 mL</td>
			<td>Schott AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>Carl Roth GmbH &#38; Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MillexGV Filter (5 &#181;m)</td>
			<td>Merck Millopore Ltd., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needler holder</td>
			<td>Geuder AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR International, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Sarstedt AG &#38; Co., Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel (single use), triangular blade</td>
			<td>Aesculap AG &#38; Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette 10 mL</td>
			<td>Sarstedt AG &#38; Co., Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette 5 mL</td>
			<td>Sarstedt AG &#38; Co., Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cups</td>
			<td>Greiner Bio-One, &#214;sterreich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Paul Hartmann AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical drape</td>
			<td>Paul Hartmann AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stitch scissors</td>
			<td>Geuder AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Ethilon 10-0 Polyamid 6</td>
			<td>Ethicon Inc., USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (5 mL)</td>
			<td>Becton Dickinson, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>trephine &#248; 7.5 mm</td>
			<td>own production</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tying forceps</td>
			<td>Geuder AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing paper</td>
			<td>neoLab Migge GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment &#38; Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular surgical microscope</td>
			<td>Carl Zeiss AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera mounted on microscope</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellSens Entry (software)</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold-light source</td>
			<td>Schott AG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heraeus GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope</td>
			<td>Olympus GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer with heating function</td>
			<td>IKA-Werke GmbH &#38; Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter pHenomenal</td>
			<td>VWR International, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop CS2</td>
			<td>Adobe Systems, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision scale</td>
			<td>Ohaus Europe GmbH, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a porcine corneal endothelial organ culture model using split corneal buttons

角膜内皮细胞的实验研究与几个困难有关。供体角膜很少，很少可用于实验性调查，因为通常移植需要角膜。内皮细胞培养物往往不能很好地翻译到体内的情况。由于非人类角膜的生物结构特征，栽培过程中的基质肿胀会导致角膜内皮细胞大量流失，长期难以进行栽培。脱毛剂（如 dextran）用于抵消此响应。然而，它们也导致显著的内皮细胞损失。因此，建立了不需要脱肿剂的体外器官培养模型。当地屠宰场的猪眼被用来准备分裂的角膜纽扣。局部角膜发角膜后，角膜的外层（表皮、弓形层、频闪部分）被移除。这大大减少了角膜内皮细胞损失引起的大规模基质肿胀和Descemet膜折叠在较长的培养期，并改善内皮细胞层的一般保存。随后，完全角膜裂解，然后从剩余的眼球和培养中去除分裂的角膜按钮。使用光显微镜在制备后长达15天的随访时间（即第1天、第8天、第15天）评估内皮细胞密度。使用制备技术可以更好地保存内皮细胞层，通过较少的基质组织肿胀实现，从而导致分裂角膜按钮与人类供体角膜相媲美的缓慢和线性下降率。由于这种标准化的有机体典型栽培研究模式首次允许稳定种植至少两周，它是人类供体角膜的宝贵替代品，用于今后研究各种外部因素，对角膜内皮的影响。

提出的解剖技术意味着部分去除基质组织，导致角膜样本更薄，从而减少基质肿胀（图1和图2）。较少基质肿胀诱导较少的剪切和捏力，对角膜内皮有负面影响，从而导致较低的内皮细胞损失率6。与非分裂的角膜纽扣和整个角膜样本相比，分离角膜按钮在15天的培养后明显具有更好的保存性内皮细胞层，这反?...

Watch this Scientific Journal Video about A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons at JoVE.com

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

这里提出了猪分裂角膜纽扣的制备和培养的分步方案。由于这种通常培养的器官培养模型显示细胞死亡率在15天内，相当于人类捐赠角膜，它代表了第一个模型，允许长期种植非人类角膜，而不添加有毒的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 ...

该协议提出了第一个研究模型，允许组织性长期培养角膜内皮细胞通过准备分裂角膜按钮实现。通过部分去除外角膜层，内皮细胞损失显著减少，允许角膜内皮细胞的培养至少15天。因此，该模型对于研究特定物质或感兴趣的技术（如灌溉溶液或眼膜粘弹性装置）对角膜内皮的影响具有价值。在死后接受被移除的猪眼后，使用眼剪刀和大肠杆菌钳切除所有轨道副眼镜。将五到六只眼睛放入五%的碘PBS溶液中，共5分钟，以适当消毒眼表面，并小心每分钟搅拌一次，确保表面完全浸入水中，实现完全消毒。五分钟后，将消毒的眼睛转移到一杯纯PBS上，并小心搅拌，从眼表面彻底清洗碘PBS溶液。在开始解剖之前，使用钝导管将五毫升注射器填充两毫升刚解冻的胎儿小腿血清，并通过 0.22 微米过滤器将血清应变成 80 毫升无脱酸培养剂。轻轻搅拌混合物，实现血清的均匀分布，并在12井细胞培养板的每个井中加入三毫升的培养基。当板准备就绪时，将眼球转移到眼科手术显微镜下的眼球支架上，角膜朝上，并使用充满 0.9% 氯化钠的注射器将轻微的吸力吸到眼球支架上，将眼睛固定到位。使用含有标准化镶嵌的颤音，表面切入中央角膜，不超过300微米，并使用大肠杆菌钳和一个带三角形刀片的一次使用手术刀，通过频闪水平切割和去除角膜的部分颤音片段。在实验过程中，将10-0缝合线放在频闪中，使内皮与频闪侧分化，无需穿透角膜内皮，并使用无镶嵌的颤管将切入至全深度，直到到达前眼室。在角膜完全穿透后，可以察觉前眼室的电阻和液体泄漏明显下降。将获得的分离角膜按钮（包含频闪的一部分、Descemet 膜和角膜内皮）放入 12 孔板的一个孔中，带标签的一侧朝下。然后在培养盘上标上每个井，每个分裂的角膜按钮都有单独的编号，以便进行后续检查时进行识别。收集所有角膜按钮后，将12井板放在细胞培养培养箱中，温度为37摄氏度，二氧化碳含量为5%，湿度为95%，如果超过7天的孵化期，则在第8天更换上经剂。要进行未污染的检查，在12井板的每个井中加入三毫升的低度平衡盐溶液，并小心地将一个分裂的角膜按钮放入每一个井中，以诱导角膜内皮细胞肿胀，并提高细胞计数的可见性。一到两分钟后，使用显微镜相机获取内皮三个不同区域的至少三张图像，以对角膜内皮的实际状况进行有代表性的印象，并添加一个真实到刻度长度为 100 微米的刻度栏，供以后分析。为防止渗透损伤，在最多五分钟后将分离角膜按钮从溶液中转移回培养基。要进行染色检查，请将分裂的角膜按钮放在单独的 Petri 盘中，内皮侧向上，并使用移液器缓慢地将 0.25% Trypam 蓝色溶液滴到每个角膜内皮上，每个试样 90 秒。接下来，在使用移液器将0.2%的Alizarin Red S溶液缓慢地滴到内分，90秒后，在0.9%的氯化钠中小心地冲洗按钮。然后像刚才演示的一样对样本进行图像显示。要为细胞密度和形态评估准备图像，请打开适当的图形编辑软件程序中的图像，并在图像上投射一个真实到刻度的方长为 100 微米。要裁剪比例图条，请使用矩形选框工具与比例图条边框并选择编辑和裁剪。单击文件和新的，然后选择剪贴板。记下其他图片的相应像素，然后单击"确定"。再次单击文件和 new，然后根据比例栏的长度在弹出窗口中插入计数正方形的宽度和长度（以像素为单位）。选择白色作为背景颜色，然后单击"确定"以选择全部，然后复制所选图像。选择角膜内皮的图像，将正方形粘贴到角膜内皮上。选择方形图层，将不透明度设置为 30%，然后单击"确定"，然后用 100 微米的幻灯片长度将投影的正方形保存图像。要评估内皮细胞密度，请打开 ImageJ 中投影的正方形的图像，然后打开单元格计数器插件。要计算正方形中的单元格，请单击初始化以计算正方形两侧的内皮单元格，并记录结果。要评估形态参数，计算每个细胞边界内至少四个细胞的联合会议、特征玫瑰花环形状的外观以及投影方块内的阿里扎林红区。在15天期间，分裂的角膜按钮表明内皮细胞密度稳步下降，第15天内皮细胞层的平均每周百分比内皮细胞损失为4.90%，因此可以对角膜细胞进行形态分析，改造数据约占合并细胞边界的7%，每毫米样本的中位数约为每毫米一个玫瑰形成，每毫米平方大约 13 个正时电池损耗。在这里，在低氧平衡盐溶液的微观评估中，以及用锥蓝和阿里扎林红S染色后，可以观察到角膜内皮的代表性图像。如果分裂的角膜按钮被培养与内皮侧朝下，广泛的内皮细胞损伤是预料之中的。非分裂角膜按钮因频闪肿胀而显著增加内皮细胞损失，导致Descemet的膜在15天的培养中折叠，而分裂角膜纽扣在15天的培养后表现出大部分保存的角膜内皮，这从分散的守时Alizarin红染色区域就证明，表明单一的细胞被破坏。为了尽量减少细胞损失，在该协议的任何步骤中，应注意不要接触角膜内皮。

Research

JoVE Journal

Medicine

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

角膜共聚焦显微镜：一种新型的非侵入性技术来量化小纤维病变周围神经病变

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

科研

医学

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

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 ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

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, ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

使用Active波尔图腔静脉分流术猪肝脏采购和原位移植技术

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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 ...

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

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 ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

使用24小时器官保存和连续遥测的猪模型中正交肾自动移植

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...