Simple 3D-printed stirred bioreactor enhances retinal organoid production via improved oxygenation.

Static cultures, the standard for growing monolayer cells, were presumed suitable for and widely used during the initial adherent phase of retinal organoid (RO) differentiation. This assumption may be incorrect, as monolayer human pluripotent stem cells (hPSCs) become highly structured and multilayered during neural induction. These emerging complex structures likely consume more oxygen than passive diffusion can provide. To test this hypothesis, we measured oxygen at the vessel bottom and found levels fell below 1%, indicating non-physiological hypoxia. This condition contributed to cellular necrosis and degeneration. To address this problem, we developed a 3D-printed stirred bioreactor (SBR) that maintains physiological oxygen levels. This approach significantly improved organoid yield and quality while being easily adaptable to typical laboratory workflows. We conclude that non-physiological hypoxia, a previously unappreciated condition, is a limiting factor underlying inconsistent RO yield and quality. Physiological oxygen levels can be restored by the SBR platform, resulting in greater consistency and improved outcomes.