(B-67) A Novel Tissue Bioreactor for Retinal Organoid Microenvironmental Control

Introduction::
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 (Fig. 1A). Here we developed a novel tissue bioreactor allowing the maturation of inner and outer retinal cell phenotypes within an O₂ gradient.




Materials and Methods::
The bioreactor is assembled from a 75x25x3 mm acrylic slide, a gas-permeable film, a cover glass, and double-sided adhesives, which were adjusted with computer numerical control milling and laser cutting (Fig. 1B). The 60 culture wells of 1.5 mm in diameter and 0.7 mm high each hold one retinal organoid. A sodium sulfite solution provides the bioreactor with an oxygen leaching channel and a dual syringe pump creates a 10 µL/min continuous flow of culture medium though the bioreactor (Fig. 1C). Gas diffusion through the gas permeable membrane and culture medium was predicted using computational modeling software for atmosphere (20.9% O₂) and incubator (18.6% O₂) conditions. O₂ concentration measurements were performed with O₂ sensors along the z-axis in 50 µm steps in atmospheric conditions.




Results, Conclusions, and Discussions:: The gas diffusion throughout the culture medium resulted in an O₂ concentration gradient along the z-axis. (Fig. 1D). The computational predictions in atmospheric conditions are in accordance with measurements around the retinal organoid location in the bioreactor.

This open-well bioreactor is easily accessible for downstream analysis, establishes a steep O₂ 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 O₂ concentration gradient.



Acknowledgements (Optional): : This project is supported by NIH/NCI grant R01 CA248890 (DP)


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