Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus. Given the complexity of this disease and the limitations of the existing in vitro and in vivo models, there is a great need to develop alternative models that are useful for elucidating DR mechanisms and testing novel active compounds. Therefore, this study aimed to establish an innovative ex vivo model of DR. To investigate the effects of promising factors for DR treatment, such as recombinant human nerve growth factor (rhNGF) and human amniotic fluid stem cells (hAFSCs), we employed human corneal lenticule (hCL), a discarded ocular tissue obtained from refractive procedure, as delivery system. To this end, porcine retinal explants were exposed to high glucose (HG; 25 mM for 4 days) and then co-cultured with hCL engineered with rhNGF and hAFSCs. The results showed that exposure to HG led to an increase in the gene expression of inflammatory, oxidative stress, and apoptotic markers (GFAP, NF-kB, NRF2, NOS2, BAX/Bcl-2, and p21) and reduced the expression of retinal cell structural markers (TUBB3 and Rho). Increased inflammation was also confirmed by cytokine array experiments performed on HG-treated retinal explant-conditioned medium. Interestingly, co-culture with hCL engineered with rhNGF and hAFSCs reduced in HG-treated retinal explants the expression of inflammatory, apoptotic, oxidative markers and the level of pro-inflammatory cytokines while enhancing IL-10 levels and retinal structural markers. Overall, our data suggest that porcine retinas treated with HG could be a valuable model for replicating the DR milieu. Additionally, our findings indicate that rhNGF and hAFSCs delivered by hCL hold promise as therapeutic strategies for mitigating the effects of DR.