Retinal Organoids Long-Term Functional Characterization Using Two-Photon Fluorescence Lifetime and Hyperspectral...

Area of Science:

• Stem cell biology
• Regenerative medicine
• Ophthalmology

Background:

• Pluripotent stem cell-derived organoids offer preclinical and therapeutic potential.
• Challenges in organoid research include heterogeneous yields and subjective selection, impacting reproducibility.
• Quality control is crucial for reliable organoid-based studies.

Purpose of the Study:

• To introduce a live imaging technique for non-invasive, long-term monitoring of retinal organoids (RtOgs).
• To characterize metabolic and structural changes during RtOg development using advanced microscopy.
• To enhance the quality control and repeatability of organoid experiments.

Main Methods:

• Utilized two-photon microscopy for live imaging of RtOg development.
• Employed fluorescence lifetime imaging microscopy (FLIM) to assess metabolic trajectories (free/bound NADH ratio).
• Applied hyperspectral imaging for structural and molecular characterization, validated by qPCR, scRNA-seq, and immunohistochemistry.

Main Results:

• Identified a metabolic shift from glycolysis to oxidative phosphorylation between months 2-3 of differentiation.
• Observed photoreceptor gene expression emerging around month 2, correlating with metabolic changes.
• Demonstrated that RtOg cellular composition and lamination at 3-6 months mimic in vivo development.

Conclusions:

• Live imaging, including FLIM and hyperspectral imaging, provides robust quality control for RtOg development.
• Metabolic and molecular profiles correlate with distinct developmental stages in RtOgs.
• This technique enhances the consistency and reliability of stem cell-derived organoids for research and therapy.