Uchida Tsutomu, Furukawa Maho, Kikawada Takahiro, Yamazaki Kenji, Gohara Kazutoshi
Division of Applied Physics, Faculty of Engineering, Hokkaido University, N13 W8 Kita-ku, Sapporo, Hokkaido 060-8628, Japan.
Anhydrobiosis Research Group, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan; Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8561, Japan.
Cryobiology. 2017 Aug;77:50-57. doi: 10.1016/j.cryobiol.2017.05.008. Epub 2017 May 25.
Trehalose is a promising natural cryoprotectant, but its cryoprotective effect is limited due to difficulties in transmembrane transport. Thus, expressing the trehalose transporter TRET1 on various mammalian cells may yield more trehalose applications. In this study, we ran comparative cryopreservation experiments between the TRET1-expressing CHO-K1 cells (CHO-TRET1) and the CHO-K1 cells transfected with an empty vector (CHO-vector). The experiments involve freezing under various trehalose concentrations in an extracellular medium. The freeze-thawing viabilities of CHO-TRET1 cells are higher than those of CHO-vector cells for most freezing conditions. This result differs from control experiments with a transmembrane type cryoprotectant, dimethyl sulfoxide (MeSO), which had similar viabilities in each condition for both cell types. We conclude that the trehalose loaded into the cells with TRET1 significantly improves the cryoprotective effect. The higher viabilities occurred when the extracellular trehalose concentration exceeded 200 mM, with 250-500 mM being optimal, and a cooling rate below 30 K/min, with 5-20 K/min being optimal.
海藻糖是一种很有前景的天然冷冻保护剂，但其冷冻保护作用因跨膜运输困难而受到限制。因此，在各种哺乳动物细胞上表达海藻糖转运体TRET1可能会带来更多海藻糖的应用。在本研究中，我们对表达TRET1的CHO-K1细胞（CHO-TRET1）和转染空载体的CHO-K1细胞（CHO-载体）进行了比较冷冻保存实验。实验包括在细胞外培养基中不同海藻糖浓度下进行冷冻。在大多数冷冻条件下，CHO-TRET1细胞的冻融活力高于CHO-载体细胞。这一结果与使用跨膜型冷冻保护剂二甲基亚砜（MeSO）的对照实验不同，在每种条件下两种细胞类型的活力相似。我们得出结论，通过TRET1加载到细胞内的海藻糖显著提高了冷冻保护效果。当细胞外海藻糖浓度超过200 mM时活力更高，250 - 500 mM为最佳浓度，冷却速率低于30 K/min时活力更高，5 - 20 K/min为最佳冷却速率。
