Open-End Control of Neurite Outgrowth Lengths with Steep Bending Confinement Microchannel Patterns for Miswiring-Free Neuronal Network Formation

Wiring technology to control the length and direction of neurite outgrowth and to connect them is one of the most crucial development issues for forming single-cell-based neuronal networks. However, with current neurite wiring technology, it has been difficult to stop neurite extension at a specific length and connect it to other neurites without causing miswiring due to over-extension. Here, we examined a novel method of wiring neurites without miswiring by controlling the length of neurites in open-ended bending microchannel arrays connected beyond the maximum bending angle of neurite outgrowth. First, we determined the maximum bending angle of neurite elongation to pass through the bending point of a bending microfluidic channel; the maximum angle (the critical angle) was 90 degrees. Next, we confirmed the control of neurite outgrowth length in open-ended microchannels connected at 120 degrees, an angle beyond the maximum bending angle. The neurites stopped when elongated to the bend point, and no further elongation was observed. Finally, we observed that in bending microchannel arrays connected at an angle of 120 degrees, two neurite outgrowths stopped and contacted each other without crossing over the bend point. The results show that the steep bending connection pattern is a robust open-end neurite wiring technique that prevents over-extension and miswiring.