Center for Engineering in Medicine and BioMEMS Resource Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Cryobiology. 2012 Apr;64(2):91-6. doi: 10.1016/j.cryobiol.2011.11.007. Epub 2011 Dec 3.
Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.
干燥保存作为一种节能的替代方法已经被探索出来，用于替代冷冻保存，但哺乳动物细胞对干燥应激的高度敏感性一直是将细胞储存在干燥状态下的主要障碍之一。一种降低干燥敏感性的重要策略涉及使用二糖海藻糖。当海藻糖存在于细胞膜的两侧时，已知它可以提高哺乳动物细胞的干燥耐受性。由于海藻糖是膜不可渗透的，因此涉及这种有前途的糖的干燥策略的发展受到阻碍。我们探索了使用来自非洲摇蚊 Polypedilum vanderplanki 的高容量海藻糖转运蛋白 (TRET1) 的潜力[21]，将海藻糖引入哺乳动物细胞的细胞质中，从而提高干燥耐受性。当中国仓鼠卵巢细胞 (CHO) 稳定转染 TRET1（CHO-TRET1 细胞）并在 37°C 下用 0.4M 海藻糖孵育 4 小时时，与野生型 CHO 细胞相比，海藻糖摄取量增加了七倍。在进行海藻糖负载后，通过在 14%相对湿度下蒸发干燥细胞来研究干燥耐受性。在干燥至每克干重 2.60 克水后，与对照 CHO 细胞相比，CHO-TRET1 的存活率增加了 170%，生长增加了 400%（7 天后）。我们的结果表明，细胞内海藻糖对赋予部分干燥耐受性有益。
