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Ischemia-reperfusion (I/R) injury is inevitable during free tissue transfers. When the period of ischemia exceeds the tissue tolerance, it causes necrosis and flap failure. The aim of this study was to investigate the effects of adipose-derived stem cells (ASCs) embedded in a collagen type I scaffold on the survival of free skin flaps to counteract I/R injury.Left superficial caudal epigastric skin flaps (3 × 6 cm) were performed in 28 Wistar rats that were divided into four groups. The flaps elevated in the animals of the control group did not suffer any ischemic insult, and the vascular pedicle was not cut. All other flaps were subjected to 8 hours of ischemia prior to revascularization: I/R control group (8 hours of ischemia), I/R scaffold group (8 hours of ischemia + collagen type I scaffold), and I/R scaffold-ASCs group (8 hours of ischemia + collagen type I scaffold with rat ASCs embedded). Transit-time ultrasound blood flow measurements were performed. After 7 days, the areas of flap survival were measured and tissues were stained with hematoxylin/eosin and Masson's trichrome stain for histological analysis.The mean percentage flap survival area was significantly higher in the ASCs-treated flaps (I/R scaffold-ASCs group) compared with the ischemic controls (I/R control group and I/R scaffold group). Higher vascular proliferation and lower severity of necrosis and inflammatory changes were seen histologically in the samples of the ASCs-treated group. No significant difference in blood flow was detected between groups.Subcutaneous administration of ASCs embedded on a collagen type I scaffold reduces tissue damage after I/R injury in microvascular free flaps.