Effects of Trehalose\nand Antifreeze Glycoproteins\non Long-Term Storage of Cryopreserved Trehalose-Transporter Expressing\nCells and on Ice Recrystallization

Trehalose\nas a cryoprotectant has been studied by using cells that\nexpress a trehalose transporter (TRET1). In this study, we examine\nthe freeze–thaw cycle survival fraction (viability) of cells\nusing the trehalose-TRET1 system during one year of storage at 193,\n213, and 235 K. To examine the possible influence of ice recrystallization\non viability during such storage, we also observe the crystal grain\nsizes of thin ice sample. Both sets of experiments are run with and\nwithout the antifreeze glycoprotein (AFGP) as an ice-recrystallization\ninhibiting (IRI) agent as well as with and without the trehalose.\nWithout the trehalose, we find that no cryopreservation occurs even\nwith added AFGP, indicating that AFGP is not a cryoprotectant under\nthe experimental conditions. In contrast, the viability with trehalose\nremains high for 1 year at 193 K. However, at the two higher temperatures,\nthe viability with trehalose decreases rapidly at least in the first\n2 months. In this initial period, ice recrystallization occurs even\nwhen AFGP is added, although AFGP exhibits IRI activity. This indicates\nthat both intra- and extracellular ice grow in this period. As a result,\nwe argue that the viability decrease is caused by intracellular recrystallization,\nmainly during the first 2 months of storage.