Development of an innervated human skin equivalent to model nociceptive circuitry in vitro

The reconstruction of innervated skin equivalents in vitro to recapitulate the somatosensory system is central to advancing our understanding of nociceptive circuitry and holds significant potential for various industrial applications. As skin-nerve crosstalk is increasingly recognized as a key element in skin physiology and nociception, the development of reliable in vitro models to evaluate the functional activity of neuroepithelial junctions is highly warranted. However, existing models often fall short in replicating the full complexity of interactions among sensory neurons, keratinocytes, fibroblasts, Schwann cells, and the extracellular matrix (ECM). In this study, we have developed an Innervated Human Skin Equivalent (IHSE), composed of a fibroblast-populated endogenous ECM enriched with human Schwann cells and topped with a fully differentiated epithelium that recapitulates basal, germinative, and keratinized layers. The IHSE was innervated using axonal projections from rat dorsal root ganglion (R-DRG) sensory neurons cultured on a high-density microelectrode array (HD-MEA). Axons emerging from the neuronal layer progressively extended through the dermal compartment and established connections with the epidermal layer, ultimately forming a well-structured neuroepithelial junction. Real-time electrophysiological recordings from the HD-MEA showed that both neuronal firing rates and the number of active microelectrodes increased as innervation progressed. By day 9, a fully developed neural network was established, featuring both free nerve endings-like structures and mature neuroepithelial junctions. Functional validation was performed by applying a drop of capsaicin solution to the apical side of the epidermis. This induced a distinct spatial and temporal electrical response as captured by the MEA, indicating activation of nociceptive terminals at the neuroepithelial junction. The electrical signal propagated to the DRG neurons on the MEA, effectively replicating the in vivo nociceptive transmission pathway. This model provides a relevant physiological platform for studying acute and chronic pain mechanisms and offers a valuable tool for the development of novel pain therapeutics.