Electroporation-based gene delivery and whole-organoid imaging in human retinal organoids.

Human retinal organoids (hRetOrg) derived from human induced pluripotent stem cells (hiPSCs) have emerged as powerful in vitro systems for studying retinal development, modeling retinal diseases, and evaluating therapeutic strategies. However, current genetic manipulation approaches, such as stable hiPSC line generation and viral transduction, are laborious and costly, offering limited spatial specificity and high variability in transgene expression. Here, we report a rapid, scalable, and spatially precise electroporation-based platform for efficient plasmid-based gene delivery in early-stage hRetOrg. Our method enables tunable and region-specific transfection of retinal progenitor cells without viral vectors or clonal selection. When coupled with resonant-scanning two-photon microscopy, this approach allows fast live cell imaging of whole organoids with subcellular resolution. Taken together, our versatile system supports high-throughput genetic manipulation and imaging in intact hRetOrg, advancing studies of human retinal development, gene function, and disease pathophysiology.