Background: Previous studies have shown that trehalose, when used alone as a cryoprotective agent (CPA), can maintain the viability of living tissue after cryopreservation and therefore may be used clinically for the future banking of human fat grafts. Objective: The purpose of this study is to determine the optimal concentration of trehalose used as a CPA for the cryopreservation of adipose aspirates in vitro. Methods: Adipose tissue was harvested with conventional liposuction from the abdomens of eight female patients and adipose aspirates were then collected from the resulting middle layer after centrifugation. A 3-cc specimen of adipose aspirates was cryopreserved using trehalose as a CPA in seven different concentrations (0.20, 0.25, 0.30, 0.35, 0.40, 0.50, and 0.75 mol/L, respectively). Cryopreservation was conducted with controlled slow cooling to -196°C and fast re-warming to 37°C, according to our established protocol. A 3-cc specimen of fresh adipose aspirates without cryopreservation served as the control. Fresh or cryopreserved adipose aspirates in each group were evaluated by viable adipocyte counts, glycerol-3-phosphate dehydrogenase (G3PDH) assay, and histology. Results: More viable adipocytes of cryopreserved adipose aspirates were found when a 0.35 mol/L concentration of trehalose was used, as compared to the other cryopreserved groups. In terms of viable adipocyte counts, there was no statistical difference between the fresh control group and the cryopreserved group with trehalose in that concentration (1.88 ± 0.61 vs. 2.4 ± 0.52 × 106/mL; P > .05). G3PDH assay was performed to assess intracellular function and showed no statistical significance in the fresh control group compared with all cryopreserved groups (all P > .05). Histologically, the basic structure of adipose tissue was adequately maintained in most of the cryopreserved groups. Conclusions: Trehalose as a CPA with a concentration of 0.35 mol/L appears to provide the optimal protection of adipose aspirates during cryopreservation. Further in vivo study will be needed to confirm these findings.
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