Long-Term Maintenance of Human Stem Cell-derived Neural Networks for Electrophysiology Studies

Understanding the electrophysiology and development of human neural networks has significant applications in drug testing, disease modeling, and fundamental neuroscience research. In-vitro models using human stem cell-derived neurons grown on microelectrode arrays (MEAs) have facilitated some early inquiries into the network properties of these cells. However, culturing viable human neural networks on MEAs beyond 3 weeks presents some challenges for studies in network electrophysiology. This has led researchers to utilize alternative methods, which are expensive, require significant technical expertise, and may limit accessibility and future translation. We present a simplified method for culturing human induced pluripotent stem cell-derived neurons and astrocytes on glass MEAs. We generated ten co-cultures that survived up to 123 days in-vitro without genetic modification or the use of non-human components. Fluorescence imaging confirmed the presence of mature neurons and electrophysiological recordings demonstrated neuronal activity as well as coordinated electrical firing (bursting) that evolved during maturation. Furthermore, we used this culturing method to investigate the impact of repeated electrical stimulation delivered during the maturation of these neural networks. Our findings suggest that the input of electrical stimulation during neuronal maturation at 11 days in-vitro results in differences in output firing rates and bursting frequency later on in development. Requiring only basic cell culturing skills, our technique makes the exploration of human neural networks more accessible and affordable for future studies in learning and memory.