Protocol for generating and using human iPSC-derived microglia-containing air-liquid-interface cortical organoid...

Area of Science:

• Neuroscience
• Immunology
• Stem Cell Biology

Background:

• Extended organoid cultures often suffer from necrotic core formation, limiting their utility for studying long-term cellular processes.
• Microglia, the resident immune cells of the brain, are crucial for neurodevelopment and disease, but their survival in traditional organoid models is challenging.

Purpose of the Study:

• To present a novel protocol for generating microglia-containing air-liquid-interface cortical organoids (MG-ALI-COs) capable of long-term culture.
• To overcome limitations of necrotic core formation and enhance microglia survival and homeostasis in 3D brain models.
• To provide a physiologically relevant platform for investigating human neuroimmune interactions.

Main Methods:

• Generation of air-liquid-interface cortical organoids (ALI-COs).
• Integration of macrophage precursors into ALI-COs to generate MG-ALI-COs.
• Maintenance and long-term culture of MG-ALI-COs.
• Experimental analyses including immunostaining, imaging, and patch-clamp electrophysiology.

Main Results:

• The protocol successfully generated long-term MG-ALI-CO cultures.
• Minimized necrotic core formation, promoting microglia survival and homeostasis.
• Demonstrated the utility of MG-ALI-COs for various experimental analyses.

Conclusions:

• The developed MG-ALI-CO model offers a robust system for studying microglia in a 3D, brain-like environment.
• This model facilitates research into human neuroimmune interactions over extended periods.
• The protocol addresses key limitations in current organoid technology for neuro-immunological studies.