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K.M. Engelsberg, K. Johansson, F. Ghosh; Porcine Full-Thickness Neuroretina In Vitro . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1605.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the survival and morphology of fetal porcine full thickness neuroretina in culture. Methods: Eleven E60 fetuses, from a normal pig were taken out by caesarian section. The 22 eyes were enucleated and immersed in C02-independent medium. The anterior segment was removed, and the neuroretina was dissected free. One complete eyecup and one dissected neuroretina was fixed immediately. The remaining 20 neuroretinas were explanted on Millicell®-PCF 0,4µm culture plate insert with the photoreceptor layer facing the membrane. The explants were kept under standard culture conditions, and were divided into 3 groups kept for 2 (n=5), 5 (n=6) or 10 (n=9) days in vitro (div). For measuring the extent of cell death in the explants, green nucleic acid (Sytox) was added to the medium 30 mins before fixation together with 4',6-diaminidin-2-phenylindoldihydrochloride (DAPI) used for cell identification. One explant from each group was prepared as a wholemount, and the remaining explants were sectioned on a cryostat, and stained with hematoxylin and eosin. Each specimens was examined in both light- and fluorescent microscope. Results: The major part of the immediately fixed neuroretina consisted of a neuroblastic layer (NBL). The ganglion cell layer (GCL) consisted of multiple rows of cells, and in the nerve fiber layer (NFL) a capillary network was seen. The GCL and the NBL were separated by the inner plexiform layer (IPL). In explants kept 2 div, the GCL was thinner, but the NBL and IPL appeared similar, except for in one specimen, where a slight folding in the inner part was seen. In 2 of the 5 examined specimens kept 5 div, no GCL could be seen, and the remaining retinas displayed only a thin rim of cells in this layer. In a few of these specimens, the NBL was separated into 2 separate cell layers by the developing outer plexiform layer (OPL). In all specimens examined after 10 div, the OPL was broader and more distinct, separating the NBL into an inner and outer nuclear layer. Three 10-day explants displayed limited folding of the innermost layers, and in 1 specimen, fully developed rosettes were seen. The remaining 2 retinas displayed a normal lamination. Wholemount prepaparations revealed extensively Sytox labeled cells in the GCL at 2 and 5 div. At 10 div less cells in this layer were labeled, but some labeled cells were found in the NBL. Conclusions:Cultured fetal porcine full-thickness neuroretina can survive and develop according to its intrinsic time table for at least 10 days in vitro. In most cases, the explants also retain the normal laminated structure. These results may be of importance in future retinal transplantation experiments.
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