Large-Scale Control of Neuronal Networks In Vitro Using Perforated Microfluidic Devices

Neurons in the brain are organized and connected into complex networks in which electrochemical signaling forms the basis for all brain function. Cortical neuronal net-works are arranged in distinct modular, layered, and hierarchical structures, underlying its diverse functions such as learning, memory, or vision. Modern biotechnology has enabled an array of techniques to culture human neural cells, ranging from discreet co-cultures to complex developmental organoids, but all of which almost exclusively form unstructured and hypersynchronous networks. Overcoming this and capturing the functional and anatomical properties of the brain in vitro has proven to be a great challenge. Current techniques for guiding neuronal connectivity in vitro is often limited to a small fraction of the total population of neural cells, leaving most of the culture effectively unguided. To provide large-scale guidance of neurons in culture, we developed a microtunnel device which allows large-scale cell entry through an array of perforations, and guides neuronal network formation through a series of tunnels. Human neural stem cells capable of forming extensive neuronal projections were used to investigate several different microtunnel designs. One particularity noteworthy design which produced predominantly unidirectional growth was used to successfully validate its effect on propagation of neural activity on microelectrode arrays. Serendipitously, we found that our microtunnels had an extraordinary effect on signal-to-noise ratio and the quality of electrophysiological recordings with regards to number of active channels and detected spikes. Since we often found the neuronal growth surprising, we developed a simple computer model which could reproduce neuronal growth in the various tunnels, allowing computer aided design (CAD) of future projects.