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Recent studies have shown potential ways for improving stem cell cryopreservation. The major need for autologous stem cell use is a long-term storage: this arises from the humans’ hope of future use of their own cells. Therefore, it is important to evaluate the cell potential of vitality and differentiation before and after cryopreservation. Although several studies have shown a long-term preservation of adipose tissue, a few of them focused their attention to stem cells. The aim of this study was to evaluate the fate of cryopreserved stem cells collected from adipose tissue and stored at low a temperature in liquid nitrogen through an optimal cryopreservation solution (using slowly cooling in 6% threalose, 4% dimethyl sulfoxide, and 10% fetal bovine serum) and to develop a novel approach to efficiently preserve adipose-derived stem cells (ASCs) for future clinical applications. Results showed that stem cells, after being thawed, are still capable of differentiation and express all surface antigens detected before storage, confirming the integrity of their biology. In particular, ASCs differentiated into adipocytes, showed diffuse positivity for PPARg and adiponectin, and were also able to differentiate into endothelial cells without addition of angiogenic factors. Therefore, ASCs can be long-term cryopreserved, and this, due to their great numbers, is an attractive tool for clinical applications as well as of impact for the derived market.