Culture media alter retinal organoid physiology promoting AAV transduction and retinal ganglion cell survival.

Human iPSC-derived retinal organoids offer a human-relevant platform for inherited retinal disease (IRD) gene therapy, yet robust AAV transduction in vitro remains challenging. Here we show that culture in BrainPhys™ (BP) medium markedly enhances AAV-mediated gene delivery. Brief BP exposure during the transduction window improves uptake, whereas continuous BP culture yields the strongest effects, indicating contributions from acute entry processes and longer-term cellular remodelling. Quantitative proteomics reveal coordinated upregulation of viral entry and trafficking mediators, including integrins that form αVβ5, and the trans-Golgi entry factor GPR108, together with enrichment of endosomal/ER-Golgi transport proteins, providing a mechanistic basis for enhanced vector processing. BP concurrently promotes neuronal maturation and metabolism, with pronounced increases in neurotrophic and synaptic proteins and broad enhancement of oxidative phosphorylation components. Functional calcium imaging demonstrates comparable neuronal dynamics across conditions but reveals robust, recurrent network-level bursts under BP, consistent with strengthened connectivity. BP also preserves retinal ganglion cells and maintains expression of canonical markers, aligning with a BDNF-centred interactome and improved circuit integration. Collectively, these findings identify the culture environment as a critical determinant of AAV efficacy in human retinal tissue models and position BP as a simple, scalable strategy to reduce vector requirements, enhance retinal cells targeting, and increase the fidelity of organoid-based non-animal testing for IRD gene therapy development.