Characterizing Tissue Oxygen Tension During Neurogenesis in Human Cerebral Organoids

Area of Science:

• Neuroscience
• Biomedical Engineering
• Biophysics

Background:

• Oxygen tension critically influences early human neurogenesis.
• Accurate, non-perturbing methods for measuring intra-tissue oxygen in 3D organoid models over extended periods are lacking.
• Current techniques like microsensors or high-excitation probes limit measurement duration or disturb organoids.

Purpose of the Study:

• To present a detailed protocol for measuring intra-organoid oxygen levels in human cerebral organoids (hCOs).
• To enable longitudinal tracking of oxygen dynamics linked to neurodevelopmental processes.
• To overcome limitations of existing oxygen measurement techniques in complex 3D biological models.

Main Methods:

• Utilized ruthenium-based CPOx microbeads embedded within human cerebral organoids (hCOs).
• Employed widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) for oxygen sensing.
• Developed an end-to-end workflow including organoid patterning, bead co-embedding, standardized FD-FLIM acquisition, automated data analysis in MATLAB, and Stern-Volmer calibration.

Main Results:

• Generated per-bead oxygen maps and longitudinal oxygen patterns within single hCOs.
• Stratified oxygen dynamics based on bead location (intra-organoid vs. gel) and sample state (healthy vs. abnormal).
• Demonstrated the ability to link developmental growth and oxygen dynamics at the bead level.

Conclusions:

• The presented protocol offers a robust, non-perturbing method for longitudinal oxygen tension measurement in hCOs.
• This technique facilitates a deeper understanding of oxygen's role in human neurogenesis and organoid development.
• The workflow provides reproducible, ready-to-use tools for researchers studying 3D organoid models.