Effects of Trehalose and Antifreeze Glycoproteins on Long-Term Storage of Cryopreserved Trehalose-Transporter Expressing Cells and on Ice Recrystallization

Trehalose as a cryoprotectant has been studied by using cells that express a trehalose transporter (TRET1). In this study, we examine the freeze–thaw cycle survival fraction (viability) of cells using the trehalose-TRET1 system during one year of storage at 193, 213, and 235 K. To examine the possible influence of ice recrystallization on viability during such storage, we also observe the crystal grain sizes of thin ice sample. Both sets of experiments are run with and without the antifreeze glycoprotein (AFGP) as an ice-recrystallization inhibiting (IRI) agent as well as with and without the trehalose. Without the trehalose, we find that no cryopreservation occurs even with added AFGP, indicating that AFGP is not a cryoprotectant under the experimental conditions. In contrast, the viability with trehalose remains high for 1 year at 193 K. However, at the two higher temperatures, the viability with trehalose decreases rapidly at least in the first 2 months. In this initial period, ice recrystallization occurs even when AFGP is added, although AFGP exhibits IRI activity. This indicates that both intra- and extracellular ice grow in this period. As a result, we argue that the viability decrease is caused by intracellular recrystallization, mainly during the first 2 months of storage.