Protective Effects and Action Mechanisms of Intracellular Trehalose on Survival of L-929 Fibroblastic Cells During Cryopreservation

Abstract

Recently, there has been much interest in using trehalose and other small carbohydrates to preserve mammalian cells in the dried state as an alternative to cryopreservation. In this study, we have aimed at testifying the hypothesis that the deleterious effects of cryopreservation of mammalian cells can be ameliorated by the addition of disaccharides, such as sucrose, maltose and trehalose, to the storage media. For this purpose, the protective effects of disaccharides on unilamella vesicles (ULVs) of dimyristoylphosphatidylcholine (DMPC) and L-929 fibroblasts were investigated by monitoring the change of ULVs diameter and assaying the cell viability, respectively. The mean diameters of ULVs with different disaccharide contents were measured by using photon correlation spectroscopy. For investigating the cryoprotective effects of intracellular disaccharides, L-929 cells were cryopreserved in complete culture media containing various concentrations (0.5, 1.0 and 2.0 mM) of sucrose, maltose or trehalose at −25oC, −75oC and −150oC during 4 d. After quick thawing at 37oC, the cellular viability was determined with water-soluble tetrazolium salt assay. Cryoprotective effects of trehalose were better than those of other disaccharides and DMSO. The mechanism of cryoprotective action of intracellular trehalose could be explained in part by the high flexibility of glycosidic bond in trehalose molecules and their interaction with phospholipid bilayers of cells. It is suggested that trehalose may be effectively applied for various fields as the storage of cell therapeutics and biological agents, the longer-term preservation of transplantable tissues and artificial organs as well as tissue engineering and regenerative medicine.