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Hsiang-Rong Tsai, Ákos Lukáts, Arnold Szabo, Christine Diane Harman, Andras M Komaromy; Organotypic Culture System for the Adult Canine Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):360.
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Organotypic retinal culture systems have been established for a number of species, including swine, rodents, rabbit, and chicken. Because of our research focus on dog models of inherited retinal diseases, the aim of this study was to develop such a culture system for the adult canine retina.
Globes were collected from two young adult wt dogs (ages: 29 and 22 wks). The neurosensory retinas were collected within 3-4 hrs post enucleation, cut into smaller pieces (2-4 mm diameter), transferred onto a semi-porous polycarbonate membrane, and kept in culture for 21 days. Between 24 and 31 retinal pieces were cultured per globe. The cell culture media contained a 1:1 mixture of Dulbecco's Modified Eagle Medium and F-12 Nutrient Mixture, supplemented with vitamins, amino acids, and hormones. Every 2 days, retinal pieces were fixed and processed for frozen sections and flat-mounts, followed by cell-specific immunolabeling for cones (hCAR, L/M-, and S-opsin), rods (rhodopsin), bipolar (G0alpha), and glial cells (glutamine synthetase and GFAP).
The adult canine retina could be maintained with our organotypic culture system for the entire 21-day study period with all cell types evaluated remaining viable. Progressive degenerative changes were observed within the photoreceptors, mostly at the 2- and 3-wk time points. These included gradual loss of outer and inner segments and mislocalization of opsins. Sustained upregulation of GFAP expression with formation of new glial processes was seen in both astrocytes and Müller cells starting within the first few days in culture.
This is the first successful attempt of an organotypic culture system for the adult canine retina. The integrity of the photoreceptors was best preserved within the first 7 days. This may represent a valuable tool for detailed examination of disease mechanisms and preliminary evaluation of new therapies in mutant retinas.
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