Decoding Pain: Next-Generation In Vitro Systems for Mechanistic Insights and Drug Discovery

Chronic pain affects approximately 20% of the population, significantly impacting daily life and increasing psychosocial burden for patients due to the limited effect of analgesics in providing reliable pain relief. This clinical impediment is largely due to a limited mechanistic understanding of human pain pathophysiology, caused by the limitation of models to study human pain mechanisms. Further, the lack of reliable models to study human pain-associated mechanisms hinders the screening and evaluation of pain-related drugs and therapies, leading to significant obstacles in the development of pain medications without inducing unwanted side effects. More complex and physiologically relevant in vitro models provide an opportunity to study human cells and tissues in a controlled environment while replicating key aspects of the native human environment. Further, these models are ethically advantageous by serving the 3R principle and enable the direct study of human cells and their physiological environments, facilitating the development of translational findings. In this review, we present the key molecular mechanisms of the pain sensory process, highlight the bidirectional crosstalk between nociceptors and non-neuronal cells at the peripheral and central nervous system levels, discuss the current in vivo models and their drawbacks, and explore strategies for human-relevant modeling by generating human nociceptors in vitro through various differentiation protocols of induced pluripotent stem cells (iPSCs). We also review the state-of-the-art of in vitro pain model systems, including their electrophysiological characterization, compartmentalization strategies, and the use of agonist and antagonist assays targeting specific ion channels and receptors to validate these models. Additionally, we examine pain coculture model strategies that more closely replicate in vivo peripheral and central microenvironments. Finally, we discuss the current limitations and future perspectives of enhancing the physiological relevance and predictability of in vitro pain models for the development of novel analgesics and deepening mechanistic understanding.