The establishment of the presented research model was supported by KMU-innovativ (FKZ: 13GW0037F) of the Federal Ministry of Education and Research Germany.

This protocol follows the ethical guidelines of our institution. In accordance with the statutes of our institution&#39;s ethical review committee no ethical approval had to be obtained prior to the experiments, as all porcine corneas were obtained from the local slaughterhouse.
1. Organ culture
<ol>
	<li>Prepare pig eyes.
	<ol>
		<li>From the local slaughterhouse, obtain pig eyes that were removed shortly postmortem but before thermal treatment. Transport the eyes to the lab and process the...

<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. M., Kantelip, B. M., Delbosc, B. Y., Oppermann, M. T., Laroche, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology,+Histology+and+Ultrastructure+of+Human+C31+Organ-Cultured+Corneas.">Morphology, Histology and Ultrastructure of Human C31 Organ-Cultured Corneas.</a> Cornea. 14 (3), 300-310 (1995).</li><li>Linke, S. 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>

This protocol provides a method for the preparation of porcine split corneal buttons, which represents a standardized and low-cost ex vivo corneal endothelial organ culture model for research purposes6. Porcine split corneal buttons showed a decrease of the endothelial cell density comparable to endothelial cell losses observed in human donor corneas cultivated in eye banks over a two-week period6,10,11<s...

Corneal transplantation procedures are among the most commonly performed transplantations worldwide1. As there is a severe shortage of human donor corneas, experimental research addressing corneal endothelial cells in human corneas is difficult to perform1. However, the introduction of irrigation solutions and other substances used within the eye, ophthalmic viscoelastic devices, as well as surgical instruments and techniques (e.g., phacoemulsification instruments and techniques, ultrasound energy) requires valid and extensive investigations regarding their effects on the corneal endothelium before clinical use.
Few alternatives to human donor corneas exist for research. Animal research models are very valuable but at the same time very resource consuming and increasingly questioned ethically. A major drawback of in vitro cell cultures is their limited translation to the human eye. Results obtained from cell cultures can be incongruous to in vivo conditions, because cells may undergo endothelial mesenchymal transition (EMT), resulting in fibroblast-like morphology caused by the loss of cell polarity and changes in cell shape and gene expression2.
Whereas previous ex vivo models reported cultivation periods of up to only 120 h, a novel preparation technique to establish a porcine corneal endothelial organ culture model by culturing fresh pig corneas for at least 15 days was recently introduced3,4,5,6. If the corneal epithelium and parts of the stroma are removed (approximately 300 &#181;m in total) from the cornea prior to cultivation, swelling of the stroma is reduced in split corneal buttons resulting in less endothelial cell loss and a well maintained endothelial cell layer after up to 15 days, whereas non-split corneal buttons show significant endothelial cell loss due to uneven stromal swelling and formation of Descemet's folds. Eye banks usually use osmotic deswelling agents such as dextran to reduce swelling of corneas prior to transplantation. However, these agents were shown to induce increased endothelial cell loss7,8,9.
This article aims to visualize this standardized ex vivo research model in a detailed step-by-step protocol in order to enable future investigators to perform research on the corneal endothelium using split corneal buttons. This model represents a straightforward method to test substances and techniques used within the eye, such as ophthalmic viscoelastic devices, irrigation solutions, and ultrasound energy, or other procedures where the corneal endothelium is of interest.

<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>
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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.

The presented dissection technique implies partial removal of stromal tissue, resulting in a thinner cornea sample and thus less stromal swelling (Figure 1 and Figure 2). Less stromal swelling induces less shear and pinch forces that have a negative impact on the corneal endothelium, thus causing lower endothelial cell loss rates6. Split corneal buttons show a significantly better-preserved endothelial cel...

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A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

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.

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

This protocol presents the first research model allowing organotypical long-term cultivation of corneal endothelial cells achieved with the preparation of split corneal buttons. By partially removing the outer corneal layers, the endothelial cell loss is significantly reduced, allowing the cultivation of corneal endothelial cells for at least 15 days. Therefore, this model is valuable for investigating the effects of specific substances or techniques of interest such as irrigation solutions or ophthalmic viscoelastic devices on the corneal endothelium.After receiving pig eyes that were removed shortly post-mortem, use eye scissors and Colibri forceps to remove all of the orbital adnexes. Place five to six eyes into five percent iodine PBS solution for a total of five minutes to properly disinfect the eye surfaces, with careful stirring once a minute to ensure that the surfaces are fully submerged to achieve a complete disinfection. After five minutes, transfer the disinfected eyes to a cup of pure PBS, and carefully stir it to thoroughly wash the iodine PBS solution from the eye surfaces.Before beginning the dissection, use a blunt catheter to fill a five-milliliter syringe with two milliliters of freshly thawed fetal calf serum, and strain the serum through a 0.22 micrometer filter into 80 milliliters of dextran-free culture medium. Gently agitate the mixture to achieve a homogenous distribution of the serum, and add three milliliters of the medium to each well of a 12-well cell culture plate. When the plate is ready, transfer an eye bulb into an eye bulb holder under an ophthalmic surgical microscope with the cornea facing up, and use a syringe filled with 0.9%sodium chloride to apply slight suction to the eye over the eye bulb holder to fix the eye in place.Use a trephine containing a standardized inlay to cut superficially into the central cornea, no deeper than 300 micrometers, and use Colibri forceps and a single-use scalpel with a triangular blade to cut and remove the partially trephined fragment of the cornea horizontally through the stroma. Superficially place a 10-0 suture into the stroma to allow differentiation of the endothelial from the stromal side during the experiments, without penetrating the corneal endothelium, and use a trephine without an inlay to advance the trephine cut to the full depth until the anterior eye chamber is reached. A distinct drop in resistance and fluid leaking from the anterior eye chamber can be perceived after complete penetration of the cornea.Place the obtained split corneal button containing part of the stroma, the Descemet's membrane, and the corneal endothelium into one well of the 12-well plate with the tagged side facing down. Then label each well on the culture plate with an individual number for every split corneal button for identification during follow-ups. When all of the corneal buttons have been collected, place the 12-well plate in a cell culture incubator at 37 degrees celsius, five percent carbon dioxide, and 95%humidity, replacing the supernatant on day eight if a seven-day incubation period is exceeded.To perform an unstained examination, add three milliliters of hypotonic balanced salt solution to each well of a 12-well plate, and carefully place a single split corneal button into each well to induce swelling of the corneal endothelial cells, and to improve the visibility of the cells for counting. After one to two minutes, use a microscope camera to acquire at least three images of three different areas of the endothelium to obtain a representative impression of the actual condition of the corneal endothelium, and add a scale bar with the true-to-scale length of 100 micrometers for later analysis. To prevent osmotic damage, transfer the split corneal button from the solution after a maximum of five minutes back into the culture medium.To perform a stained examination, place the split corneal buttons in individual Petri dishes, endothelial side up, and use a pipette to slowly drop 0.25%Trypam blue solution onto each corneal endothelium for 90 seconds per specimen. Next, carefully rinse the buttons three times in 0.9%sodium chloride in a small glass beaker before using a pipette to slowly drop 0.2%Alizarin Red S solution onto the endothelia for 90 seconds. Then image the samples as just demonstrated.To prepare the images for cell density and morphology assessment, open the images in an appropriate graphics editing software program and project a square with a true-to-scale side length of 100 micrometers on the image. To crop the scale bar, use the rectangular marquee tool to border the scale bar and select edit and crop. Click file and new, and select clipboard.Note the corresponding pixels for the other pictures and click OK.Click file and new again, and insert the width and length of the counting square in pixels in the pop-up window according to the length of the scale bar. Select white as a background color, and click OK to select all, and copy the selected image. Select the image of the corneal endothelium and paste the square on the corneal endothelium.Select the square layer and set the opacity to 30%Then click OK and save the image with the projected square with the true-to-scale slide length of 100 micrometers. To assess the endothelial cell density, open the images with the projected squares in ImageJ, and open the cell counter plugin. To count the cells within the square, click initialize to count the endothelial cells on two sides of the square, and record the result.To assess the morphological parameters, count the joint meeting of at least four cells per cell border, the characteristic rosette-shaped appearance, and the Alizarin Red areas within the projected squares. Over a period of 15 days, split corneal buttons demonstrate a steady decline in the endothelial cell density, with an average weekly percentage endothelial cell loss of 4.90%Staining of the endothelial cell layer on day 15 allows morphological analysis of the corneal cells, with reformation figures making up approximately seven percent of the merging cell borders, a median of approximately one rosette formation per millimeter squared per sample, and about 13 punctual cell losses per millimeter squared. Here, representative images of the corneal endothelium during microscopic evaluation in hypotonic balanced salt solution, and after staining with Trypan blue and Alizarin Red S, can be observed.If the split corneal buttons are cultivated with the endothelial side facing down, extensive endothelial cell damage is to be expected. Non-split corneal buttons suffer significantly increased endothelial cell loss due to stromal swelling, causing Descemet's membrane folding over 15 days of cultivation, whereas split corneal buttons exhibit a largely preserved corneal endothelium after 15 days of cultivation, as evidenced by scattered punctual Alizarin Red-stained areas, indicative of single destroyed cells. To minimize cell loss, care should be taken to not touch the corneal endothelium throughout any steps of this protocol.

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7.0K ビュー.  University Medical Center Hamburg-Eppendorf (UKE). このプロトコルは、分割角膜ボタンの調製により達成された角膜内皮細胞の有機的な長期培養を可能にする最初の研究モデルを提示する。外側角膜層を部分的に除去することにより、内皮細胞損失が著しく減少し、角膜内皮細胞の培養を少なくとも15日間可能にする。したがって、このモデルは、特定の物質や、角膜内皮に対する灌漑溶液や眼用粘弾性デバイスなどの目...