Introduction: Vascular Composite Allograft (VCA) transplantation involves tissue including skin and muscle. Clinical VCA is complicated by limited ex vivo storage times. This study evaluates the effect of Necrostatin-1 in preventing cell death of VCA relevant skin and muscle cell lines exposed to prolonged cold storage followed by rewarming.
Methods: Rat skin cell fibroblast cell line (RSF) and skeletal muscle cell line (L6) were cultured in 24-well plates with or without coverslips. Cells maintained at 37°C in complete media were used as controls. Cells subjected to cold storage were placed in cold saline with or without 150µM of RIPK1 inhibitor, Necrostatin-1, for 24 hours at 4°C. Cells subjected to cold storage followed by rewarming were placed in complete media at 37°C for 24 hours following cold storage. Flow cytometry with Annexin V (AV) and Propidium iodide (PI) staining was used to determine cell viability (AV-PI-), and the percentage of cells that have undergone early apoptosis (AV+PI-), late apoptosis/necroptosis (AV+PI+) and necrosis (AV-PI+). RSF and L6 cells subjected to cold storage with or without Necrostatin-1, and cells subjected to cold storage with or without Necrostatin-1 followed by rewarming, were examined for the presence of  pro-caspase 3 and cleaved caspase 3 by immunofluorescence.
Results: Necrostatin-1 treatment decreased the percentage of early apoptotic (AV+PI-), late apoptotic/necroptotic (AV+PI+), and necrotic (AV-PI+) cells, and increased the percentage of viable cells in skin RSF cells exposed to both cold storage and cold storage followed by rewarming (Figure 1, left). In muscle L6 cells exposed to cold storage alone, Necrostatin-1 improved cell viability by reducing early and late apoptosis/necroptosis, and necrosis. In L6 cells exposed to cold storage followed by rewarming, the necrotic (AV+PI+) population was notably decreased with Necrostatin-1 treatment (Figure 1, right).
Cleaved caspase-3 expression was increased in RSF and L6 cells exposed in cold storage followed by rewarming, compared with control cells. Treatment with Necrostatin-1 markedly reduced the cleaved caspase-3 staining in both RSF and L6 cell lines exposed to cold storage and rewarming (Figure 2). 
Conclusion: Necrostatin-1 improved skin and muscle cell viability in vitro by inhibiting both apoptosis and necrosis. These results suggest that the addition of Necrostatin-1 to organ preservation solutions may prolong VCA storage times for transplantation. Further studies to confirm these findings in a rat hind limb transplantation model are ongoing.