Abstract
Hair follicle cells reside within a complex extracellular matrix (ECM) environment in vivo, where physical and chemical cues regulate their behavior. The ECM is crucial for hair follicle development and regeneration, particularly through epithelial-mesenchymal interactions. Current in vitro models often fail to replicate this complexity, leading to inconsistencies in evaluating hair loss treatments. Advanced 3-dimensional (3D) culture systems that better mimic in vivo ECM dynamics are needed for more effective therapeutic assessments. Here, we introduce a 3D co-culture system designed to replicate in vivo ECM dynamics. The system incorporates primary dermal papilla cells from human patients, co-cultured with neonatal keratinocytes. This platform facilitates uniform spheroid formation through cell sliding and aggregation, enabling the evaluation of approximately 60 spheroids per well. The model is optimized for high-throughput screening, allowing precise assessments of hair-loss-inducing compounds under consistent conditions. We successfully generated dermal papilla cell and keratinocyte spheroids that closely resemble the native ECM structure, providing an optimal microenvironment for studying hair follicle biology. The 3D co-culture model supported efficient spheroid formation with consistent cellular organization and polarization, along with enhanced ECM-related gene expression crucial for hair follicle regeneration. Uniform spheroid formation and reproducibility were demonstrated across experiments. Overall, the novel 3D co-culture system provides a robust platform for replicating in vivo-like ECM conditions, enabling effective assessment of potential hair loss treatments through epithelial-mesenchymal interactions. Its high-throughput capacity, combined with reproducibility and ease of use, makes it a valuable tool for screening therapeutic candidates and advancing hair loss treatment development.