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...

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

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 ...

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JoVE Journal

Medicine

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