UC Irvine

Retinal degeneration (RD) is a leading cause of vision impairment and blindness worldwide and treatment for advanced RD does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Besides, subjective tissue selection reduces the repeatability of organoid-based scientific experiments and clinical studies. Controlled and predictable methodology and tools are needed to standardize RtOg production, characterization and long-term maintenance.To optimize and standardize RtOg long-term maintenance, we designed a shear stress-free micro-millifluidic bioreactor platform for nearly labor-free retinal organoid maintenance. We compared different 3D printers for fabricating the mold from which Polydimethylsiloxane (PDMS) was cast to create the bioreactor. We optimized the bioreactor design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity. Once assembled, we successfully cultured RtOgs using different designs of the bioreactors for up to 4 months. We used different quantitative and qualitative techniques to characterize the RtOgs produced with our bioreactors and compared with those produced with conventional culture to demonstrate the superiority of our approach. At the same time, to improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy (2PM) to non-invasively monitor RtOgs’ long-term development. Fluorescence Lifetime Imaging Microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging (HSpec) was applied to characterize structural and molecular changes. These live imaging experimental results were confirmed with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. In addition, we applied an advanced electrophysiology testing system to further verify the functionality of matured RtOgs cultured in the bioreactor platform. Spontaneous and light-stimulated spiking activities were observed. In summary, we designed and optimized a bioreactor for long term RtOg culture in a low shear stress environment that was also compatible with multimodal imaging. We have demonstrated a 2PM-based non-invasive imaging technique to monitor RtOg metabolic and structural changes at the cellular level throughout the entire differentiation and development process. The health of mature RtOgs were further verified with electrophysiological measurements. The methodology and the findings of this study are of great value in live RtOgs long-term maintenance, characterization and monitoring, offering potentially powerful tools in screening and quality control for RtOg production.