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

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. 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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. 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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>
</table>

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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Research

JoVE Journal

Medicine

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.

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

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

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

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

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

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

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

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

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

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

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

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 injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

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

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.

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

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).

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

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

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

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.

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 ...