Abstract
Purpose :
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. Here we developed a novel tissue bioreactor allowing the maturation of inner and outer retinal cell phenotypes within an O2 gradient.
Methods :
The bioreactor is assembled from a 75x25x3 mm acrylic slide, a PFA film, a cover glass, and double-sided adhesives, which were adjusted with computer numerical control milling and laser cutting (Fig. 1A). The 60 culture wells of 2 mm in diameter and 0.7 mm high each hold one retinal organoid. A nitrogen (N2) tank provides the bioreactor with 5 mL/min N2 gas and a dual syringe pump creates a 5 µL/min continuous flow of culture medium though the bioreactor (Fig. 1B). Gas diffusion through the PFA 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 :
The gas diffusion throughout the culture medium resulted in an O2 concentration gradient along the z-axis (Fig 1C). The computational predictions in atmospheric conditions are in accordance with the measurements around the retinal organoid location in the bioreactor (Fig. 1D).
Conclusions :
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.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.