Trehalose uptake and dehydration effects on the cryoprotection of CHO-K1 cells expressing TRET1

Chinese hamster ovary cells (CHO-K1 cells) in which the trehalose transporter (TRET1) is expressed can have greater cryoprotection than ordinary CHO-K1 cells. This study examines the uptake characteristics of trehalose into cells via TRET1 and determines the influence of intracellular trehalose on the freeze-thaw viabilities. In our experiments, the intracellular trehalose concentration is controlled by the extracellular trehalose concentration and the immersion time in a freezing solution. In this freezing solution, both kinds of CHO-K1 cells are independently dispersed with various amount of trehalose, and then put into the CO₂ incubator for 0-6 h. After a set immersion time, the cell-suspended sample is cooled to 193 K, stored for 1 week, then quickly thawed at 310 K and its viability measured. The uptake amount of intracellular trehalose is measured before freezing. We find an upper limit for the uptake amount of trehalose when the extracellular trehalose concentration is about 400 mM, at which the freeze-thaw viability is the highest. When the extracellular trehalose concentration exceeds 400 mM, shorter immersion times are needed to obtain the maximum freeze-thaw viability. Also, longer immersion weakens the cells. Our analyses indicate that when the extracellular trehalose-concentration is less than 400 mM, the trehalose uptake occurs more slowly with less dehydration, resulting in less stress on the cell. When the extracellular trehalose concentration exceeds the saturation level, the cell is stressed by the excess dehydration due to the remaining osmotic pressure, with apoptosis occurring before freezing.