Abstract
In-vitro culture systems generally apply homogeneous stimuli and rely on intercellular signaling to guide growth of tissues. However, to derive complex tissue structures such as the human retina, a gradation of certain stimuli is required. The inner retina resides in a hypoxic environment (2% O2) adjacent to the vitreous cavity. From there, oxygenation levels rapidly increase towards the outer retina (18% O2) at the choroid. We developed a retinal organoid chip allowing the maturation of inner and outer retinal cell phenotypes. The chip is assembled from a micro-milled acrylic slide, a gas-permeable film, a cover glass, and double-sided adhesives. The 55 culture wells, connected by a singular channel, each hold one retinal organoid. A sodium sulfite solution provides the chip with an oxygen leaching channel and a push/pull syringe pump creates a 50 µL/hour continuous flow of culture medium though the bioreactor. Retinal organoids are placed in the wells exposed to the oxygen gradient for the duration of 20 weeks. The gas diffusion throughout the culture medium resulted in an oxygen concentration gradient along the z-axis. Retinal organoids are successfully differentiated and present both inner and outer retinal cell phenotypes. This open-well retinal organoid chip is easily accessible for downstream analysis, establishes a steep oxygen gradient and allows high-throughput retinal organoid culture. It will help retinal organoids mature into the complex structure to use them for disease modeling and drug testing. Furthermore, the culture system contains a holder setup to facilitate the use of microfluidics during culture and allows transport to a microscope for live imaging. Continuous culture medium flow provides a stable environment without changes in pH due to waste secretion of organoids. In addition to retinal organoid culture, this bioreactor can also be used for other applications that benefit from an oxygen concentration gradient.