Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.

毛囊形态发生是由毛囊胚芽（HFG）上皮和间充质层之间的相互作用引起的。在这里，我们开发了HFGs大规模制备方法在体外通过细胞的自组织。我们在悬浮液中混合了小鼠表皮细胞和小鼠/人间充质细胞，并将它们接种在定制设计的阵列板的微孔中。在三天的培养期间，细胞最初形成随机分布的单细胞聚集体，然后在空间上彼此分离，表现出典型的HFG形态特征。这些自分选的毛囊胚芽（ssHFGs）被证明能够在皮内移植到裸鼠的背部时有效地产生毛囊和产生轴。这一发现促进了在由透氧有机硅制成的微孔阵列芯片中大规模制备约5000 ssHFG。我们证明了通过硅树脂芯片底部的氧气供应的完整性对于实现ssHFG的形成和随后的发干的形成至关重要。最后，将芯片上空间对齐的ssHFGs封装到水凝胶中，并同时移植到裸鼠的背部皮肤中，以保留其居间空间，从而产生空间对齐的毛囊。这种简单的ssHFG制备方法是改善当前头发再生医学技术的一种有前途的策略。