Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany.
Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Hannover, Germany.
Sci Rep. 2017 Jul 24;7(1):6198. doi: 10.1038/s41598-017-06542-z.
The aim of this study was to investigate preservation of biomolecular structures, particularly DNA, in freeze-dried fibroblasts, after loading with trehalose via freezing-induced uptake. Cells were freeze-dried with trehalose alone or in a mixture of albumin and trehalose. Albumin was added to increase the glass transition temperature and storage stability. No viable cells were recovered after freeze-drying and rehydration. FTIR studies showed that membrane phase behavior of freeze-dried cells resembles that of fresh cells. However, one day after rehydration membrane phase separation was observed, irrespective of the presence or absence of trehalose during freeze-drying. Freeze-drying did not affect the overall protein secondary structure. Analysis of DNA damage via single cell gel electrophoresis ('comet assay') showed that DNA damage progressively increased with storage duration and temperature. DNA damage was prevented during storage at 4 °C. It is shown that trehalose reduces DNA damage during storage, whereas addition of albumin did not seem to have an additional protective effect on storage stability (i.e. DNA integrity) despite the fact that albumin increased the glass transition temperature. Taken together, DNA in freeze-dried somatic cells can be preserved using trehalose as protectant and storage at or below 4 °C.
本研究旨在探讨通过冷冻诱导摄取将海藻糖载入冻干成纤维细胞后，生物分子结构（特别是 DNA）的保存情况。细胞分别用海藻糖、白蛋白和海藻糖的混合物进行冻干。加入白蛋白是为了提高玻璃化转变温度和储存稳定性。冻干和复水后没有可存活的细胞回收。傅里叶变换红外光谱研究表明，冻干细胞的膜相行为类似于新鲜细胞。然而，复水后一天，无论在冻干过程中是否存在海藻糖，均观察到膜相分离。冻干不会影响蛋白质的整体二级结构。通过单细胞凝胶电泳（“彗星试验”）分析 DNA 损伤表明，DNA 损伤随储存时间和温度的延长而逐渐增加。在 4°C 下储存可防止 DNA 损伤。研究表明，海藻糖可减少储存过程中的 DNA 损伤，而添加白蛋白似乎对储存稳定性（即 DNA 完整性）没有额外的保护作用，尽管白蛋白提高了玻璃化转变温度。综上所述，使用海藻糖作为保护剂，将 DNA 保存在 4°C 或以下，可以保存冻干体细胞中的 DNA。
