A Study Of In Vitro Preservation Of Human Hair Follicle

BackgroundThe in vitro preservation of hair follicles has great significance to clinical treatment of hair transplantation. Currently, cryopreservation is the most widely investigated method for the in vitro preservation of human hair follicles but still dissatisfies the clinical needs. Thus, to explore a more effective cryopreservation agent for the preservation of hair follicles is very important. On the other hand, cells residing in skin have complicated interactions with the hair follicle and influence its development. Cells-based tissue engineered skin may provide a physiological environment similar to native tissue. In this study, we employed the tissue engineering technology as a new way to preserve the hair follicle at room temperature in vitro for the first time.Objective1. To improve the viabilities of the human hair follicles stored by vitrification through adding different cytokines.2. To store hair follicles at room temperature by inserting hair follicle units into tissue engineered skin.Methods1. After dissecting scalp into single undamaged hair follicle, the follicles were randomly divided into different groups for routine culture, slow freezing, vitrification without cytokine and vitrification with different cytokines. The hair follicles were cryopreserved for24hours,72hours,1month, and6months respectively, and were detected by length measuring, HE, and immunofluorescent staining.2. Hair follicle units were inserted to the engineered dermis constructed by fibroblasts and rat tail tendon collagen type I. Then, the keratinocytes were implanted for three times. After1week of submerged culture and3weeks of air-liquid culture, tissue engineered skin and the hair follicles were detected by inverted microscope, HE, and immunohistochemical staining.Results1. After cryopreservation for different stages, the recovered hair follicles in the vitrified group with cytokine HGF showed a better viability and a higher growth rate than that in the other cryopreservative groups, and no significant difference compared to that in the routine culture group. HE staining results confirmed that in the cytokine HGF group more cells were alive and the follicle constitution was more complete than that in the other cryopreservative groups. And immunofluorescence staining showed that there were more hair follicle stem cells in the cytokine HGF group.2. After4weeks of in vitro construction, skin appendages such as hair follicle and sebaceous glands were found in the group of engineered skin with hair follicles. And the complete hair follicles could be maintained for more than21days without hair papilla damage, which was longer than those of free-floating culture. In the group of engineered skin with hair follicles, a stratified and cornified epidermis formed with positive expression of typical differentiation markers of Keratin4and Keratin10, and showed a continuous basement membrane with positive staining of Laminin and Collagen IV.ConclusionCompared with other methods, the optimized cryopreservation agent with cytokine HGF can improve the viability of hair follicle after vitrification. And the new hairy human engineered skin model has shown that the morphology of hair follicles can be maintained longer, meanwhile the engineered epidermis has differentiated better. These two storage methods for hair follicles can provide new strategies for both hair transplantation surgery and tissue engineered skin construction.