High-Pressure Skin Graft Preservation

Briefly, C57BL6J/Jcl mice (CLEA Japan Inc., Tokyo, Japan) were euthanized by carbon dioxide inhalation and their skin backs were resected. Then, 8-mm punch biopsy tools (Kai Industries Ltd., Gifu, Japan) were used to obtain full-thickness skin grafts (total of 80 skin grafts). Ten grafts were packed into each of 25-ml cryopreservation bottles (Perfluoroalkoxy [PFA] bottle; Tech-jam, Osaka, Japan) filled with DMEM solution (Nissui Pharmaceutical Co.). Skin grafts in each bottle were pressurized at 50 MPa for 36 h using a new automated HHP device as mentioned above or at 200 MPa for 10 min using another custom-made device (Echigo Seika Co.), as described in our previous studies^{11 (link),30 (link)} (Fig. 1b). After pressurization, those specimens were washed 2 times with PBS and transplanted to the dorsal fascia of other mice (2 grafts per mouse, total 12 grafts per group). We then covered the grafts with collagen sheets and closed the skin by nylon 5-0 sutures (Fig. 1b).