Simple and efficient method for consecutive inactivation-cryopreservation of porcine skin grafts

We previously reported that inactivation treatment by high hydrostatic pressurization (HHP) has potential utility as a novel skin regeneration therapy for various skin tumors. In this study, we evaluated whether glycerol-cryopreservation could be applied in order to preserve inactivated skin by HHP using a porcine model. Twenty full-thickness skin grafts (1.5 × 1.5 cm) were prepared from a minipig. The skin samples were inactivated by the HHP in normal saline or glycerol/fructose solution, followed by cryopreservation for 5 weeks at - 80 °C in each same solution. Another 10 grafts immediately after inactivation were prepared as non-cryopreserved controls. Nine grafts in each group were randomly implanted on the fascia of a host pig and removed at 1, 4 and 11 weeks after grafting. All grafts showed engraftment macroscopically. Hematoxylin eosin staining showed the cellular components in all areas of the dermis at 4 and 11 weeks after grafting, and immunohistochemical staining for CD31 showed the presence of capillaries in the grafts in all groups. The surface and cross-sectional areas of grafts in the normal saline solution cryopreserved group decreased between 1 and 11 weeks, whereas these areas in the glycerol cryopreserved group did not decrease significantly. Glycerol cryopreservation may therefore be a simple and efficient method for preserving porcine skin inactivated by HHP.