Optimizing the in vitro neuronal microenvironment to mitigate phototoxicity in live-cell imaging

Area of Science:

• Neuroscience
• Cell Biology
• Biotechnology

Background:

• Long-term live imaging of neuronal networks is crucial for understanding development.
• Fluorescent imaging techniques often cause phototoxicity, impacting cell survival and network formation.
• Optimizing culture conditions is essential to mitigate phototoxicity and improve imaging quality.

Purpose of the Study:

• To develop and optimize a live-imaging protocol for long-term neuronal network formation in vitro.
• To quantitatively analyze the impact of different culturing conditions on neuronal morphological health and viability.
• To identify optimal conditions for minimizing phototoxicity during live imaging of neuronal cultures.

Main Methods:

• A human embryonic stem cell-derived cortical neuron reporter line was used.
• Neurons were cultured in 8 different microenvironments varying in extracellular matrix (human vs. murine laminin), culture media (Neurobasal™ vs. Brainphys™ Imaging media), and seeding density.
• Quantitative analysis included PrestoBlue viability assays, digital PCR for gene quantification, and an automated image analysis pipeline for network morphology.
• Daily fluorescent imaging was performed for 33 days.

Main Results:

• Brainphys™ Imaging medium significantly supported neuron viability, outgrowth, and self-organization compared to Neurobasal™ medium.
• The combination of Neurobasal™ medium and human laminin reduced cell survival.
• Higher seeding density promoted somata clustering but did not significantly increase viability.
• A synergistic effect between culture media and laminin type was observed, with Brainphys™ Imaging medium showing protective properties.

Conclusions:

• Brainphys™ Imaging medium offers superior support for neuronal viability and network formation in live-imaging protocols compared to Neurobasal™ medium.
• Culture media composition, particularly the presence of light-protective compounds, plays a critical role in mitigating phototoxicity.
• Optimized culture conditions, including media choice and extracellular matrix, are vital for successful long-term live imaging of neuronal networks.