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Arnold Szabo, Anna Enzsoly, Klaudia Szabo, Agoston Szel, Ákos Lukáts; Long term organotypic culture of the human retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2981. doi: https://doi.org/.
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Corneal transplantation has become a routine procedure in ophthalmology nowadays. The anterior segment with the cornea of the donor eyeball is removed shortly before transplantation, and the otherwise intact posterior segment containing the retina that could be used for scientific investigations is usually disposed of. Here we present a potent in vitro application of these retinas that allows the long term survival of retinal cells with maintained retinal integrity.
Approximately 5x5 mm pieces of central retina were harvested from donor adult eyes 2-4 hours after enucleation. The pieces of the neural retina without the pigment epithelium were placed onto a nitrocellulose membrane and kept in culture using serum-free medium for up to three weeks. The cultures were fixed at different time points, processed and analyzed as histological sections or whole mounts by immunohistochemistry using cell type-specific antibodies.
Although slight degenerative signs were present, the overall retinal architecture was well preserved even after 21 days in culture and the retinal thickness was comparable to that of in vivo controls. As expected, we detected signs of glial activation with only moderate GFAP immunoreactivity after 7 days in culture which gradually increased thereafter. The most prominent alterations occurred in the outer and inner plexiform layers, where reduction of thickness and minor synaptic disorganization was observed. The cones showed a near normal morphology with occasional swelling of the outer segments. The rod outer segments gave a strong anti-rhodopsin immunoreaction, but they were shorter than in in vivo controls. Some picnotic nuclei in the outer and inner nuclear layers were detectable after prolonged culturing, but no severe decrease in the number of bipolar, amacrine and horizontal cells was found.
Our results indicate that the adult human retina can be maintained in an appropriate culture system for at least three weeks. Despite the minor changes detected, the retina shows normal lamination, the retinal cells preserve their normal morphology and staining characteristics. The presented culture method can be reliably reproduced and could be adapted to pharmacological and toxicological applications.
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