Cryopreservation of tissue-engineered epithelial sheets in trehalose

Tissue-engineered epidermal membranes are useful for clinical wound healing. To facilitate these products in the clinic, optimized storage methods need to be developed. We studied the efficiency of extracellular trehalose at various concentrations for cryopreserving human tissue-engineered epidermal membranes compared with that of dimethyl-sulfoxide (DMSO) used by most organ banks for cryopreserving skin grafts and artificial skin substitutes. Keratinocyte (KC) viability, proliferation and marker expression following cryopreservation in trehalose were examined with similar results to those using DMSO. Trehalose concentration (0.4m) was optimized according to the described cellular activities following cryopreservation. Artificial epidermal substitutes were then cryopreserved in trehalose at the optimized concentration. Cell viability, growth factor secretion and wound healing properties of cryopreserved artificial epidermal substitutes using nude mice were examined and compared with those of DMSO cryopreservation. Cryopreservation with trehalose enhanced human KC viability in suspension and artificial skin substitutes. In addition, trehalose cryopreservation provided fast recovery of EGF and TGF-β1 secretion by KCs after thawing. When transplanted into nude mice, trehalose-cryopreserved artificial skin repaired skin defects in a similar manner to that of a non-cryopreserved control. Moreover, trehalose-cryopreserved artificial skin resulted in engraftment and wound closure that was significantly enhanced compared with that of DMSO-cryopreserved epidermal membranes. The results indicate that the use of trehalose improves cryopreservation of tissue-engineered epithelial sheets.