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Mohit Mohit, Nathan Breuillard, Nina Harmening, Thais Bascuas Castillo, Alain Conti, Gregg Sealy, Gabriele Thumann, Martina Kropp; Up-scaling and humanization of retinal organotypic culture as a model for intraocular efficiency and toxicity analyses.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3161 – F0435.
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© ARVO (1962-2015); The Authors (2016-present)
New intraocular drugs still need to be tested in in vivo models before becoming approved. Available retina cultures are only of limited value to replace in vivo studies due to a short life time, a small number of samples gained per retina, and mostly they are of animal origin. We aimed to prolong the duration of a retina organotypic culture, to increase the number of samples harvested per retina, and to humanize the system from pig to human retina. The enhanced model offers the analysis of efficiency and toxicity of intraocular drugs while replacing animal experiments according to 3R and being highly transferable to human patients.
Retinae derived from pig (from a local slaughterhouse 5-8h post-sacrifice) or from human donor eyes (from the Lions Gift of Sight Eyebank 2-5d post-enucleation); both were cultured in serum free Ames’ medium supplemented with growth factors (1% N2, 1% B-27) for 14d at 21°C. Retinae were analyzed for viability performing a PI staining, a CytoTox-Glo® and a TUNEL assay. Morphology was assessed in flatmounts by a customized score (0=no damage to 3=severe damage, cell loss) and in cross sections after histological staining, while cell-type-specific degradation and inflammation were evaluated by immunofluorescent staining (rhodopsin, GFAP, Iba-1).
The combination of hypothermia, cell-protective growth factors, optimized tissue processing, and the harvest of 6 mm small punch-samples (12-24 samples/retina) preserved retinal structure for 14d in pig and human cultures as quantified by the modestly increasing score from 0.56 to 1.91 (pig) and 1 to 1.95 (human) (0 vs. 14d); and qualitatively assessed in H&E-stained sections. Viability decreased moderately over time (0 vs. 14d: 20.6 vs. 39.1 mean grey value [PI+ cells], 2690 vs. 2002 AU [LDH-release]; 2.5 vs. 135 apoptotic cells/section). Immunohistology confirmed presence of photoreceptors up to day 11 and a low activation of Muller and microglia cells in both, pig and human retina.
Summarized, pig and human neural retinas (central & peripheral parts) can be cultured for 14d preserving key cell types and structure. Degradation, cell loss and inflammatory cell activation are modest, allowing efficiency and toxicity analyses. The enhanced model is suitable to evaluate the benefit of new intraocular treatments while using less animals and improving transferability to humans.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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