Due to an increasing number of patients affected by age-related neurodegenerative diseases, there is a pressure to develop new effective drugs to treat the latter. However, new drugs still need to be tested in-vivo before being approved by regulatory authorities because of a lack of suitable model systems, though a reduction of animal testing is desired. Purpose of the present project was to enhance the ex-vivo retina organotypic culture system, i.e., to prolong the duration of the culture, to increase the number of samples harvested per retina, and to humanize the system from pig to human retina in compliance with the 3R principles. Adult porcine or human donor neural retinas were isolated and cultured in static conditions for 14 d in serum free Ames’ medium supplemented with growth supplements at 21⁰C with the photoreceptor layer facing downwards. To determine retinal viability, propidium iodide (PI) staining (necrosis), CytoTox-Glo, and a TUNEL assay (apoptosis) were performed. Morphology of retinal cells and tissue layers were assessed by a customized degeneration score and in cross sections after hematoxylin and eosin (H&E) staining. Changes in retinal thickness were evaluated by measuring the total thickness and the thickness of individual cell layers of the retina in H&E-stained sections. Rhodopsin, GFAP, Iba-1 and vimentin immunofluorescence staining were used to analyze cell-type-specific degradation and inflammation. The combination of hypothermia, cell-protective growth factors, optimized tissue processing, and the harvest of 6 mm small punch-samples preserved retinal structure for 14 d in pig and human cultures as quantified by an overtime only modestly increasing degeneration. Retinal laminar structure was preserved along with thickness of retina in both, porcine and human explants for 14 d as confirmed by H&E staining. Necrosis and apoptosis increased over time but moderately. Photoreceptors could be partly preserved for 14 d in both, human and porcine retina with low activation of muller cells and microglia. The results presented here show that our static organotypic retina culture method could be significantly improved by a preservation of the retinal laminar structure, photoreceptor preservation with low level of inflammation throughout a culture duration of 14 d. It was possible to increase the sample number per retina from 4 to 32. The enhanced model is suitable to evaluate the benefit of new intraocular treatments while using less animals and improving transferability to humans.