Expansion Culture of Hair Follicle Stem Cells through Uniform Aggregation in Microwell Array Devices

[Image: see text] Hair regeneration using hair follicle stem cells (HFSCs) and dermal papilla cells is a promising approach for the treatment of alopecia. One of the challenges faced in this approach is the quantitative expansion of HFSCs while maintaining their hair induction capacity. In this study, HFSC expansion was achieved through the formation of uniform-diameter cell aggregates that were subsequently encapsulated in Matrigel. We designed a microwell array device, wherein mouse HFSCs were seeded, allowed to form loosely packed aggregates for an hour, and then embedded in Matrigel. Quantitative analysis revealed a 20-fold increase in HFSC number in 2 weeks through this culture device. Gene expression of trichogenic stem cell markers in the device-grown cells showed a significant increase compared with that of typical flat substrate Matrigel suspension culture cells. These microwell array-cultured HFSCs mixed with freshly isolated embryonic mesenchymal cells indicated vigorous hair regeneration on the skin of nude mice. Furthermore, we examined the feasibility of this approach for the expansion of human HFSCs from androgenetic alopecia patients and found that the ratio of CD200(+) cells was improved significantly in comparison with that of cells cultured in a typical culture dish or in a Matrigel suspension culture on a flat substrate. Therefore, the novel approach proposed in this study may be useful for HFSC expansion in hair regenerative medicine.