1Neurology and Neurosciences, University of Medicine and Dentistry of New Jersey - UMDNJ, 2Institute of Ophthalmology and Visual Science, University of Medicine and Dentistry of New Jersey - UMDNJ
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Wang, J., Kolomeyer, A. M., Zarbin, M. A., Townes-Anderson, E. Organotypic Culture of Full-thickness Adult Porcine Retina. J. Vis. Exp. (49), e2655, doi:10.3791/2655 (2011).
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.
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 μm, 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 to create 3 legs (4 mm in height) under a ring (2 mm in height), which raised the height of the inserts by approximately 5 mm.
2. Preparation of sterile filter paper for attachment and culture of porcine retina
Whatman 4M Filter paper (Cat No. 1004) was cut into a circular shape (6.3 mm in diameter) with a small, triangular handle that was used for moving the filter paper into a glass tube for sterilization or once the retina was attached (as shown in the video).
3. Preparation of retinal explants
Eyes from 5- to 9-month old, 150-230 lbs, American Yorkshire pigs were obtained from a local abattoir within three hours of enucleation (transported on ice). Upon arrival, eyes were cleaned of extraneous tissue, dipped in betadine solution (10% Povidone-iodine, The Purdue Frederique Company, Stamford, CT) and washed twice in Dulbecco’s Modified Eagle Medium (DMEM with 1g/L glucose, L-glutamine, and sodium pyruvate, Cellgro-mediatech Inc., Manasses, VA) supplemented with 2.5 μg/ml Amphotericin B (Gibco-Invitrogen, Carlsbad, CA). The anterior segment was removed, exposing the neural retina. Six-mm trephine blades (Storz Ophthalmic-Bausch and Lomb, Manchester, MO) were used to cut equatorial, full-thickness posterior segment explants. The retina was subsequently peeled off by gently applying a piece of dry sterile filter paper onto the ganglion cell layer, lifting off the neural retina, and placing the filter paper with attached retina onto the culture insert, photoreceptors facing up. The insert was then placed into culture medium, making sure to fully cover the retina, in a 12-well culture dish (Fisher). Multiple explants were obtained routinely and cultured from a single eye.
4. Tissue culture
The retinal tissue was cultured in Eagle’s Minimum Essential Medium (MEM, Invitrogen-Gibco-Life Technologies Inc., Rockville, MD), pH 7.4, supplemented with 0.2 mg/ml glutamine, 10 μg/ml porcine insulin, 1 mM pyruvate, 0.1 mM L-ascorbic acid, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B (Antibiotic Antimycotic: Sigma, St. Louis, MO), and aerated with humidified 5% CO2, balance air, at 37 °C. Medium was exchanged every two days.
Tissue can be used, treated with a variety of reagents or probes, or left untreated for subsequent biochemical or morphological analyses. The section below describes procedures to create consistent full-thickness retinal cross-sections.
Use any standard protocol. Immunolabeled thick sections can be viewed with a confocal microscope.
7. Representative Results:
Morphology was preserved during the seven-day period of culture. Images of retina before and after culturing are available in the video.
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 photoreceptors was ameliorated by raising the height of the culture insert and agitating the culture medium. They successfully maintained adult retinal tissue for up to six days4,5. Kobuch et al. described a perfusion culture system for adult porcine retina and retinal pigment epithelium (RPE) and were able to maintain the organotypic tissue for at least 10 days7. However, the procedure for preparing the retina-RPE-choroid sheets for laboratory manipulation was complicated and required special tools. In addition, each explant required its own perfusion system making in vitro culture of multiple tissue samples cumbersome. Kaempf et al. successfully created another organotypic culture model of adult mammalian neurosensory retina in co-culture with RPE but only showed results for a 3-day culture; the long-term survival of the tissue was not tested8.
In this manuscript, we describe a simple and reproducible protocol for organotypic culture of adult porcine neural retina adopting an approach similar to that previously described for rabbit retina4 with the following features:
Given that porcine and human retina are very similar in terms of cell distribution and morphology, vascular pattern, layer thickness, and other physiologic characteristics9,10, the organotypic culture of porcine retina provides an attractive animal model for studying manifestations of human retinal diseases, degenerations, and injuries. This system may be particularly important in cases where in vitro experiments on human retina are impossible to perform due, in part, to the lack of high-quality human tissue.
No conflicts of interest declared.
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).
|5 mL pipette tip||ISC BioExpress||P-3250-19|
|NUNC cell culture insert||Thermal Scientific, Inc.||137443||8μm, Polycarbonate|
|Whatman 4M Filter paper||Whatman, GE Healthcare||1004|
|Fungizone Amphotericin B||Invitrogen||15290018|
|Trephine blades||Bausch and Lomb||T3096||6.00mm|
|O.C.T. Compound||Electron Microscopy Sciences||62550-01||4583|
|Vibratome||Leica Microsystems||Series 1000|
|Confocal microscopy;||Carl Zeiss, Inc.||LSM510|
|Anti-Synaptic Vesicle Protein 2 (SV2) mouse monoclonal antibody||DSHB||N/A|
|Anti-Glial Fibrillary Acidic Protein (GFAP) polyclonal rabbit antibody||Dako||DAKO|
|Propidium Iodide (PI)||Sigma-Aldrich||P4170|