Cryopreservation of engineered hair follicle germs for hair regenerative medicine

Abstract

Hair regenerative medicine must involve practical procedures, such as cryopreservation of tissue grafts. This can aid in evaluating tissue safety and quality, as well as transportation to a clinic and multiple transplants. Hair follicle germs (HFGs), identified during in vivo development, are considered effective tissue grafts for hair regenerative medicine. However, to the best of our knowledge, methods for cryopreserving HFGs have not been explored yet. This study investigated the efficacy of slow vitrification methods for freezing HFGs. Cryoprotectants such as dimethyl sulfoxide (DMSO) and carboxylated poly-l-lysine were used for vitrification. The results indicate that DMSO vitrification yielded the most efficient de novo hair regeneration in mouse skin, comparable to that of non-cryoprotected HFGs. A microfinger was fabricated to scale up the cryopreservation method, considering that thousands of tissue grafts were required per patient in clinical practice. The microfinger can be used for a series of processes, holding the HFG, replacing it with a cryopreservation solution, freezing it in liquid nitrogen, thawing it in a warm medium, and transplanting it into the skin. Although de novo hair regeneration by HFGs cryopreserved using microfingers was reduced by approximately 20 % compared to those cryopreserved using flat plates for fertilized eggs, it exceeded 50 %. These findings demonstrate that vitrification with DMSO and microfingers could be a useful approach for the cryopreservation of tissue grafts in hair regenerative medicine for hair loss.

Keywords: Cryopreservation; Hair follicle germ; Hair regenerative medicine; Tissue engineering; Vitrification.