Retinal organoid chip: engineering a physiomimetic oxygen gradient for optimizing long term culture of human retinal organoids

An oxygen gradient across the retina plays a crucial role in its development and function. The inner retina resides in a hypoxic environment (2% O₂) adjacent to the vitreous cavity. Oxygenation levels rapidly increase towards the outer retina (18% O₂) at the choroid. In addition to retinal stratification, oxygen levels are critical for the health of retinal ganglion cells (RGCs), which relay visual information from the retina to the brain. Human stem cell derived retinal organoids are being engineered to mimic the structure and function of human retina for applications such as disease modeling, development of therapeutics, and cell replacement therapies. However, rapid degeneration of the retinal ganglion cell layers are a common limitation of human retinal organoid platforms. We report the design of a novel retinal organoid chip (ROC) that maintains a physiologically relevant oxygen gradient and allows the maturation of inner and outer retinal cell phenotypes for human retinal organoids. Our PDMS-free ROC holds 55 individual retinal organoids that were manually seeded, cultured for extended periods (over 150 days), imaged <i>in situ</i>, and retrieved. ROC was designed from first principles of liquid and gas mass transport, and fabricated from biologically- and chemically inert materials using rapid prototyping techniques such as micromachining, laser cutting, 3D printing and bonding. After computational and experimental validation of oxygen gradients, human induced pluripotent stem cell derived retinal organoids were transferred into the ROC, differentiated, cultured and imaged within the chip. ROCs that maintained active perfusion and stable oxygen gradients were successful in inducing higher viability of RGCs within retinal organoids than static controls, or ROC without oxygen gradients. Our physiologically relevant and higher-throughput retinal organoid culture system is well suited for applications in studying developmental perturbations to primate retinogenesis, including those driven by inherited traits, fetal environmental exposure to toxic agents, or acquired by genetic mutations, such as retinoblastoma.