Skin equivalent assay: An optimized method for testing for hair growth reconstitution capacity of epidermal and dermal cells.

Hair follicle reconstitution requires highly organized epithelial-mesenchymal interactions. Skin equivalents containing the epidermal and dermal cells with hair reconstitution capacity can reproduce these processes, but have not been established. This study was conducted to develop a hair follicle-producing three-dimensional (3D) skin equivalent assay using neonate mouse epidermal and dermal cells. A skin equivalent comprised of mouse dermal cells (MDCs) embedded in type I collagen and overlaid with mouse epidermal cells (MECs) was used. MDCs were mixed with type I collagen and cultured for 7 days. One day after adding MECs on top, the composites were grafted onto nude mice. MDCs cultured on a two-dimensional (2D) plate for 7 days and mixed with MECs as a negative control, and freshly isolated MDCs and MECs mixture (chamber assay) as a positive control were also grafted. Six weeks after grafting, regenerated hair follicles were analysed. Our 3D skin equivalent culture assay reproducibly regenerated hair follicles, while MDCs precultured in the 2D model with MECs did not. Compared to the chamber assay, which produced randomly oriented hair follicles, nearly all regenerated hair follicles in our assay extruded through the skin and numerous regenerated hair follicles were higher than those in the chamber assay. Several representative genes associated with hair induction showed higher expression in our assay than in the 2D model. When Wnt3a was added, the number of regenerated hairs increased. Organized hair follicle regeneration was accomplished using our assay. This approach can be applied to assess a test agent with hair growth-promoting effects.