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Fat grafting frequently requires multiple treatments and thus repeated liposuction to achieve treatment goals. The purpose of this study was to evaluate whether cryopreservation of adipose tissue may facilitate future fat grafting.Lipoaspirates were harvested from six women and preserved using two cryopreservation methods: (1) simple cooling to −80°C (cryo-1); or (2) programmed cooling to −196°C (cryo-2). Fresh fat, cryo-1 fat, and cryo-2 fat were analyzed both in vitro and in vivo.Immunohistochemistry of both types of cryopreserved adipose tissue revealed that most adipocytes were necrotic. The cell number and viability of stromal vascular fraction cells were significantly decreased in cryo-1 fat (1.7 × 105 cells, 42.6 percent viable) and cryo-2 fat (2.0 × 105 cells, 55.4 percent viable), compared with fresh fat (3.9 × 105 cells, 90.6 percent viable). Although adipose-derived stem cells were cultured successfully from all fats, functional adipose-derived stem cells from cryopreserved fats were much fewer, with comparable multilineage differentiating capacity. In vivo studies using human fat grafted into immunocompromised mice revealed that, 3 months after transplantation, all of the cryopreserved fats maintained their volume to some extent; however, the cryopreserved fats were mostly filled with dead tissue and produced significantly lower engraftment scores than fresh fat.Most adipocytes were killed in the process of cryopreservation and thawing. Adipose-derived stem cells were isolated from cryopreserved fat, but the number of functional adipose-derived stem cells was very limited in both cryopreservation methods. After grafting, cryopreserved fat was retained as dead and fibrous tissue, suggesting a risk of clinical complications such as oil cysts.