Organotypic Culture of Full-thickness Adult Porcine Retina

Abstract

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. Protocol Making a custom-made stand for cell culture insertsThe 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. Preparation of sterile filter paper for attachment and culture of porcine retinaWhatman 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). Preparation of retinal explantsEyes 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 o...

Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems

“…In contrast, the explants cultured with the inner retina in contact with the membrane displayed better maintenance of the retinal laminar microarchitecture and significant attenuation of photoreceptor and ganglion cell death over time. Another study, by Wang et al [2011], described a nonconventional method to explant adult porcine retinas onto sterile filter paper, and it was found that retinal morphology and phenotype were wellpreserved for up to 7 days in culture. Moreover, retina explants have always been maintained at an air-liquid interface, except for the study by Wang et al [2011] in which the explant/filter paper unit was completely immersed in the cell culture medium.…”

“…Common PC inserts described in the literature come in 2 general formats: standing inserts (e.g., Millicell inserts from Milipore) or hanging inserts such as Corning's Transwell inserts. In addition to standard cell culture inserts, Wang et al [2011] built a custom-made stand using the base of a 5-mL-pipette tip to allow the circulation of culture medium to both sides of the retina. Koizumi et al [2007] also described a rabbit retina organotypic culture system, in which the oxygen supply for photoreceptors was ameliorated by raising the height of the culture insert using a custom-made stand (2 cm in diameter and 1 cm high) made from a 1-mL Monoject tuberculin syringe with agitation.…”

“…They also published a study using only the central portion of the retina corresponding to the visual streak to produce 6 small (3-4 mm 2 ) explants . Wang et al [2011] used trephine blades to generate multiple circular neuroretina explants, but did not state how many explants were produced per eye or whether they followed a quadrant isolation technique. Kuehn et al…”

“…These specimens are dehydrated in graded ethanol and embedded in paraffin wax via standard histological procedures. A few groups have also labeled the retina explant sections with Static system with elevated inserts + agitation Wang et al, 2011] Perfusion culture system [Kobuch et al, 2008] Retinal sheet (most common)…”

“…Posterior segment explant: retina, retinal pigment epithelium, choroid, sclera [Kaempf et al, 2008;Denk et al, 2015] Vitreoretinal explant [Peynshaert et al, 2017] Inner retina or GCL facing up (most common) GCL facing down [Wang et al, 2011;Taylor et al, 2014] Note: Retina explants are commonly maintained at the air-liquid interface, except for the study by Wang et al [2011], which cultured the explants completely submerged in cell culture media…”