Vitrification and ultra-rapid warming of mouse skin

Around 1.1 million burn injuries in the US require medical attention annually. The definitive treatment for severe burns is skin grafting, but many patients are too sick on initial presentation to undergo autologous grafting. Instead, cryopreserved human allogeneic skin grafts are used for coverage to prevent infection and allow for patient resuscitation. Traditional skin cryopreservation uses slow cooling with 10-20% Me2SO or glycerol as cryoprotective agents (CPAs) but results in poor cell viability, which is vital for the neovascularization and recovery of wounds. Injury results from ice formation that disrupts cells and the macroscopic skin architecture. Therefore, we developed an alternative skin cryopreservation technique using ice-free vitrification paired with rapid rewarming (VR) using metal form radiofrequency (RF) heating. This study tested VR of full-thickness tail skin grafts in a mouse model. CPA (DP6) was loaded stepwise using a protocol developed by predictive diffusion modeling and verified by micro-CT and empiric testing of viability using AlamarBlue. DP6-loaded skin grafts (∼8x6mm) were vitrified and stored at -150°C until needed and rewarmed using our ultra-rapid metal form approach in an RF coil. CPA was unloaded (stepwise), and skin grafts were tested directly for viability or transplanted onto the back of syngeneic recipients. The viability of CPA-only controls was 100% (N=8), VR skin was 100% (N=7), and slow warming was 81% (N=4). The 30-day transplant result shows that the VR skin grafts demonstrated high-level engraftment (consistent with revascularization) and no necrosis (N=10) and were comparable to the control transplant group (N=10). In contrast, the injury control (N=2) and convectively warmed skin grafts (N=4) had reduced viability after rewarming and appeared pale and necrotic after transplant. These positive results show that vitrification can improve the viability, reliability, and function of donor skin for transplant to address burn therapy and other regenerative medicine applications.