Simple 3D-Printed Stirred Bioreactor Enhances Retinal Organoid Production Via Improved Oxygenation

Retinal organoids (ROs), derived from human pluripotent stem cells (hPSCs), simulate in vivo development and retinal morphology, providing a platform to study retinal development and diseases. However, current differentiation protocols often yield inconsistent results with substantial cell line and batch variability. These protocols utilize static culture methods that rely on passive oxygen diffusion to reach the vessel bottom, where adherent hPSCs initially differentiate. Static culture is standard for adherent monolayer cells and is presumed suitable for RO differentiation. We questioned this assumption given that, during differentiation, the monolayer hPSCs become highly structured and multi-layered, first as neural rosettes and then as optic vesicles (OVs). We hypothesized that the cellular oxygen consumption rate would exceed the rate of delivery via passive diffusion, particularly to inner regions of emerging OVs. To test this hypothesis, we measured dissolved oxygen concentrations at the vessel bottom and found that within hours of media change, oxygen dropped to < 1 %, a level considered non-physiologically hypoxic, which imperils cell viability. This non-physiological hypoxia caused OV degeneration, hypoxic marker expression, and necrosis. To address this problem, we developed a novel 3D-printed stirred bioreactor (SBR) that maintains physiological oxygen levels between ~4-6%. This approach significantly improved organoid yield, quality, and reproducibility while being easily adaptable to typical laboratory cell culture workflows. We conclude that non-physiological hypoxia, a previously unappreciated condition, is a limiting factor underlying inconsistent yield and quality in RO production. Physiological oxygenation levels can be restored by the SBR platform, resulting in greater consistency and improved production outcomes.