Optimized Microwell Array Device for Preparation of Hair Follicle Germ-like Aggregates

Abstract

Hair follicle morphogenesis is triggered by reciprocal interactions between epithelial and mesenchymal layers in the hair follicle germ. Techniques to create follicle germ-like structures in vitro may be useful for understanding hair follicle morphogenesis and for creating transplantable tissue for hair regenerative medicine. Microwell arrays with oxygen-permeable materials have been proposed as a promising tool for in vitro fabrication of hair follicle germ. In this study, we investigated the effects of microwell morphology and culture process on the formation and function of hair follicle germ. Microwell geometries (flat, round-bottom, pen, and cone) significantly affected the formation of hair follicle germ and the expression of hair growth-related genes. Specifically, pen-type microwells enhanced the rate of follicle germ formation by promoting cell assembly. Even in typical round-bottom wells, centrifugal filling of cells into the microwells enhanced cell assembly and follicular germ formation rates. The higher the efficiency of two cell types to form two separate follicular germ, the higher the expression of hair growth-associated gene, versican. The highest expression of versican was observed when cells were centrifugally packed in round-bottom wells. Aside from hair follicle formation, these microwell array with various shapes may be useful for producing various other organoids and tissues.