Large-scale Preparation of Hair Follicle Germs Using Bioprinting and Spontaneous Microgel Contraction

<b>Introduction: </b>Hair loss is caused by aging, drugs, and diseases, and greatly affects people's quality of life. One of the current treatments for hair loss is hair transplantation, but there is a critical limitation that only an insufficient number of hairs are sometime available for this treatment due to the progression of the alopecia. Thus, hair regenerative medicine has emerged as a novel approach, in which hair follicle stem cells are grown in culture and are used to prepare tissue grafts. Previous studies demonstrated that <i>de novo</i> hair follicles can be generated by transplanting hair follicle stem cells into nude mice. We have previously demonstrated that hair follicle germ (HFG)-like aggregates prepared using hair follicle stem cells efficiently generated <i>de novo</i> hair follicles in the back skin of nude mice¹⁾. Although the fabricated HFGs had high hair regeneration ability, considering that HFGs <i>in vivo</i> are surrounded by extracellular matrices, further improvement of hair regeneration ability of HFGs may be possible by replicating such<i> in vivo</i> extracellular matrix microenvironments. In this study, we focused on collagen, which is abundant in the skin, and fabricated HFGs containing high-density collagen and cells by contracting collagen microgels. Then, using a bioprinter, we scaled-up this approach for preparing a large number of HFGs automatically.<br/><b>Methods: </b>Two types of hair follicle stem cells, epithelial and mesenchymal cells, were isolated from the mouse embryonic skin, and were then suspended in 2.4 mg/mL of collagen gel solution, respectively. Collagen drops were placed adjacent to each other using an electromotive pipette or bioprinter to prepare collagen-containing HFGs. Changes in the microgels diameter and cell distribution in HFGs were observed during 3 days of culture. To investigate the relationship between spontaneous contraction of microgels and hair regeneration ability, a myosin II ATPase inhibitor, blebbistatin, was added to the culture medium. Collagen-containing HFGs with/without blebbistatin after 3 days of culture and no collagen-containing HFGs which were prepared by our previous approach¹⁾ were transplanted into shallow stab wounds prepared on the back of nude mice. The number of hairs generated per transplanted site was evaluated after 3 weeks of transplantation.<br/><b>Results: </b>The collagen-containing HFGs showed spontaneous contraction by cell attraction forces during 3 days of culture. The long-side diameter of collagen drops reduced from 3.2 mm to less than 0.8 mm after 3 days of culture, wherein the cell density and collagen were enriched 12 times and higher. Interestingly, the contraction was significantly inhibited in the presence of blebbistatin, and no hairs were regenerated post transplantation. However, collagen-containing HFGs without blebbistatin regenerated hair follicles and shafts post transplantation more efficiently compared to the HFGs without collagen gel. This suggested that the spontaneous contraction of cell-encapsulated microgels provided a more suitable environment for the improvement of hair regeneration ability of HFGs. Using a bioprinter, more than 1,000 HFGs were prepared within 12 minutes automatically, and the preparation efficiency and the uniformity of the collagen microgels were improved 20-fold and 2-fold, respectively.<br/><b>Conclusions</b>: We demonstrated that HFGs can be prepared through spontaneous contraction of cell-suspended collagen microgels in the culture. The cell- and collagen-dense microenvironments were suitable for the improvement of trichogenic functions. The preparation of the HFGs were automated using a bioprinter and thus scalable for preparation of a large number of HFGs. This approach may provide a promising strategy for advancing hair regenerative medicine.<br/><b>References</b>:<b> </b>1) T. Kageyama et al., <b><i>Biomaterials</i></b>, 154, 2018