A three-compartment microfluidic platform for investigating signal transmission in the human sensory pathway

Neuropathic pain remains a significant challenge due to limited understanding of sensory signal transmission mechanisms along the sensory pathway. The sensory pathway involves peripheral nociceptors in the dorsal root ganglia (DRG) that transmit signals from skin to the central nervous system via dorsal horn neurons. Current in vitro models lack the compartmentalization and resolution needed to investigate signal modulation at distinct anatomical sites along this pathway. Here, we developed a three-compartment microfluidic platform combining human induced pluripotent stem cell (iPSC)-derived sensory neurons (hiSNs) with human primary epidermal keratinocytes (HPEKs) and iPSC-derived dorsal horn neurons (hiDHNs) in a spatially organized arrangement. The platform integrates polydimethylsiloxane (PDMS) axon-guiding microstructures with high-density microelectrode arrays (HD-MEAs), enabling single-axon electrophysiological recordings and sub-cellular level stimulation. We established viable co-cultures maintained for up to six weeks and characterized spontaneous activity across all conditions. Keratinocytes increased the number of active sensory neuron axons and their firing rates, demonstrating peripheral modulation of neuronal activity. Systematic frequency-dependent electrical stimulation revealed low-pass filtering properties at sensory neuron somata, with filtering characteristics modulated by co-culture with keratinocytes. This platform enables compartment-specific investigation of signal processing in the human sensory pathway and provides a tool for studying neuropathic pain mechanisms and testing potential therapeutics.