Name: organotypic culture of full-thickness adult porcine retina
Uploaded: 2011-03-20T11:00:00
Description: Watch this Scientific Journal Video about Organotypic Culture of Full-thickness Adult Porcine Retina at JoVE.com

This work was supported by NIH grant EY 012031 (ET-A), an award from the F. M. Kirby Foundation (ET-A), Research to Prevent Blindness, Inc. (MAZ), The New Jersey Lions Eye Research Foundation (MAZ), The Eye Institute of New Jersey (MAZ), the Joseph J. and Marguerite DiSepio Retina Research Fund (MAZ), and the Janice Mitchell Vassar and Ashby Mitchell Fellowship (AMK).

1. Making a custom-made stand for cell culture inserts 
The base of a 5 ml pipette tip (ISC BioExpress, #P-3250-19) has an inner diameter of 13 mm, which perfectly matches the bottom size (12.6 mm, outer diameter) of a 10 mm (inner diameter) NUNC cell culture insert (8 &mu;m, Polycarbonate membrane, Thermal, #137443). To make the stand, the pipette was cut off near the base to produce a 6 mm long hollow cylinder. Then, pieces were removed from the side of the cylinder using a flame-heated blade ...

<ol>
	<li>Johnson, T. V., Martin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+adult+retinal+explant+organotypic+tissue+culture+system+as+an+in+vitro+intraocular+stem+cell+transplantation+model.">Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model.</a> Invest Ophthalmol Vis Sci. 49, 3503-3512 (2008).</li><li>Kretz, A., Marticke, J. K., Happold, C. J., Schmeer, C., Isenmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primary+culture+technique+of+adult+retina+for+regeneration+studies+on+adult+CNS+neurons.">A primary culture technique of adult retina for regeneration studies on adult CNS neurons.</a> Nat Protoc. 2, 131-140 (2007).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nat Protoc. 1, 2710-2718 (2006).</li><li>Koizumi, A., Zeck, G., Ben, Y., Masland, R. H., Jakobs, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+physiologically+functional+adult+mammalian+retinas.">Organotypic culture of physiologically functional adult mammalian retinas.</a> PLoS One. 2, e221-e221 (2007).</li><li>Lye, M. H., Jakobs, T. C., Masland, R. H., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+adult+rabbit+retina.">Organotypic culture of adult rabbit retina.</a> J Vis Exp. , (2007).</li><li>Ames, A., Li, Y. Y., Heher, E. C., Kimble, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+rabbit+retina+as+related+to+function:+high+cost+of+Na++transport.">Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.</a> J Neurosci. 12, 840-853 (1992).</li><li>Kobuch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+adult+porcine+retina+and+retinal+pigment+epithelium+in+perfusion+culture:+characterisation+of+an+organotypic+in+vitro+model.">Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: characterisation of an organotypic in vitro model.</a> Exp Eye Res. 86, 661-668 (2008).</li><li>Kaempf, S., Walter, P., Salz, A. K., Thumann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+organotypic+culture+model+of+adult+mammalian+neurosensory+retina+in+co-culture+with+retinal+pigment+epithelium.">Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.</a> J Neurosci Methods. 173, 47-58 (2008).</li><li>Guduric-Fuchs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+pig+retinal+development.">Immunohistochemical study of pig retinal development.</a> Mol Vis. 15, 1915-1928 (2009).</li><li>Chandler, M. J., Smith, P. J., Samuelson, D. A., MacKay, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+density+of+the+domestic+pig+retina.">Photoreceptor density of the domestic pig retina.</a> Vet Ophthalmol. 2, 179-184 (1999).</li></ol>

Vision researchers have established a variety of retinal culture systems, including organotypic systems, to study a variety of issues, such as retinal stem cell transplantation1, retinal regeneration2, and exogenous gene expression3-5. However, adult mammalian retina is difficult to maintain in vitro, mainly due to the high-energy demands of the photoreceptors6. Koizumi et al. described a rabbit retinal organotypic culture system in which the oxygen supply for...

<table border="1">
 <tbody>
 	<tr>
 	<td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 	<td>MEM</td>
 <td>Invitrogen-Gibco</td>
 <td>10370-021</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>5 mL pipette tip</td>
 <td>ISC BioExpress</td>
 <td>P-3250-19</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>NUNC cell culture insert</td>
 <td>Thermal</td>
 <td>137443</td>
 <td>8&mu;m, Polycarbonate</td>
 </tr>
 	<tr>
 	<td>Whatman 4M Filter paper</td>
 <td>Whatman</td>
 <td>1004</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>DMEM</td>
 <td>Cellgro-mediatech Inc</td>
 <td>10-013-CV</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Agar</td>
 <td>Fluka</td>
 <td>05040</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Fungizone Amphotericin B</td>
 <td>Invitrogen-Gibco</td>
 <td>15290018</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Trephine blades</td>
 <td>Bausch and Lomb</td>
 <td>T3096</td>
 <td>6.00mm</td>
 </tr>
 	<tr>
 	<td>O.C.T. Compound</td>
 <td>Electron Microscopy Sciences</td>
 <td>62550-01</td>
 <td>4583</td>
 </tr>
 	<tr>
 	<td>&nbsp;</td>
 <td>Sciences</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Cryostat</td>
 <td>Leica</td>
 <td>CM1900</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Vibratome</td>
 <td>Leica</td>
 <td>Series 1000</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Confocal microscopy; </td>
 <td>Carl Zeiss</td>
 <td>LSM510</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Anti-Synaptic Vesicle Protein 2 (SV2) mouse monoclonal antibody</td>
 <td>DSHB</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	<td>Anti-Glial Fibrillary Acidic Protein (GFAP) polyclonal rabbit antibody</td>
 <td>DAKO</td>
 <td>DAKO</td>
 <td>&nbsp;</td>
 </tr>
 	<tr>
 	
 <td>Propidium Iodide (PI)</td>
 <td>Sigma</td>
 <td>P4170</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

organotypic culture of full-thickness adult porcine retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Watch this Scientific Journal Video about Organotypic Culture of Full-thickness Adult Porcine Retina at JoVE.com

Organotypic Culture of Full-thickness Adult Porcine Retina

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

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