A multi-well axon array device for evaluation of stimulus responses on human sensory neural spheroid-derived axon fascicles.

Understanding the response of sensory neurons involved in pain perception to external stimuli is crucial for developing chronic pain treatments and determining pain thresholds. In the field of drug discovery, cultured neurons derived from human induced pluripotent stem cells (hiPSCs) are used to evaluate pain perception. Recently, three-dimensional axonal tissues derived from hiPSC-derived sensory neural spheroids have been constructed. These three-dimensional axonal tissues can mimic axon fascicles (AFs) found in the peripheral nervous system, making these tissues promising for cellular experiments aimed at overcoming interspecies differences. However, evaluations of physical and pharmacological stimulus responses, focusing on AF function, are insufficient. Here, we develop a multi-well axon array (MAA) device for hiPSC-derived sensory neural spheroids, which has four AFs, and simultaneously evaluate various stimulus responses in these fascicles. Using the MAA device, we assess temporal changes in spontaneous activity during the functional development of axonal fascicles and the responses to thermal and pharmacological stimuli targeting nociceptors. We also confirm that the MAA device allows for electrical stimulation of localized regions within AFs and detection of stimulation-evoked responses. These findings indicate that the MAA device is useful for evaluating the properties of stimulus-evoked responses of AFs.