Long-Term Developmental Process of the Human Cortex Revealed In Vitro by Axon-Targeted Recording Using a Microtunnel-Augmented Microelectrode Array

Objective: We aimed to develop a method for evaluating developmental changes in the synchronized activity of human induced pluripotent stem cell (hiPSC)-derived neurons without extrinsic signals from feeder astrocytes. Methods: Microelectrode arrays (MEAs) and microtunnels were fabricated with photolithography and soft lithography. hiPSCs were induced to differentiate into cortical neurons, and seeded to conventional and microtunnel MEAs. Spontaneous activity was recorded every ten days, and spiking and bursting activities were elucidated. Results: First, hiPSC-derived neurons were cultured on conventional MEAs. They formed aggregates and subsequently detached from the culture substrate. Hence, no MEAs showed spontaneous synchronized activity beyond 300 days post-induction. Next, we applied a microtunnel structure designed to keep the axons on the array. Synchronized activity was then recorded from all microtunnel MEAs by 450 days post-induction. The proportion of electrodes showing neural activity was greater than that in conventional MEAs. The activity pattern reached a steady state after approximately 330 days, which may be the maturation time of the human neuronal network. Conclusion: The use of a microtunnel MEA enables the monitoring of the long-term development of human neuronal networks of cell populations that are relatively natural given their lack of astrocyte feeders. Significance: We report a more accurate method for culturing cortical neurons differentiated from hiPSCs, validating their use in elucidating cortical development and pathogenic mechanisms in humans.