Objective: Hair follicle morphogenesis and regeneration depend on intensive but well-orchestrated interactions between epithelial and mesenchymal components.Therefore, an alternative strategy to reproduce the process of epithelial-mesenchymal interaction in vitro could use a 3D system containing appropriate cell populations.Methods: The 3D air-liquid culture system for reproducibly generating hair follicles from dissociated epithelial and dermal papilla (DP) cells combined with a collagen-chitosan scaffold is described in this study.Wnt-CM was prepared from the supernatant of Wnt1a-expressing bone marrow mesenchymal stem cells (BM-MSCs) that maintain the hair-inducing gene expression of DP cells.The collagen-chitosan scaffold cells (CCS-cells) were constructed using a two-step method by inoculating the Wnt-CM-treated DP cells and epidermal (EP) cells into the CCSs.Results: The cells in the air-liquid culture formed dermal condensates and a proliferative cell layer in vitro.The CCS-cells were able to induce hair regeneration in nude mice.Conclusions: The results demonstrate that Wnt-CM can maintain the hair induction ability of DP cells in expansion-cultures, and this approach can be used for large-scale preparation of CCS-cells in vitro to treat hair loss.