Biostabilization Laboratory, Department of Mechanical Engineering, University of Minnesota, 111 Church St. SE, Minneapolis, MN 55455, USA.
Cryobiology. 2013 Apr;66(2):176-85. doi: 10.1016/j.cryobiol.2013.01.003. Epub 2013 Jan 24.
Biorepositories worldwide collect human serum samples and store them for future research. Currently, hundreds of biorepositories across the world store human serum samples in refrigerators, freezers, or liquid nitrogen without following any specific cryopreservation protocol. This method of storage is both expensive and potentially detrimental to the biospecimens. To decrease both cost of storage and the freeze/thaw stresses, we explored the feasibility of storing archival human serum samples at non-cryogenic temperatures using isothermal vitrification. When biospecimens are vitrified, biochemical reactions can be stopped, the specimen ceases to degrade, and macromolecules can be stabilized without requiring cryogenic storage. In this study, 0.2, 0.4, or 0.8M trehalose; 0, 0.005 or 0.01M dextran; and 0 or 10% (v/v) glycerol was added to human serum samples. The samples were either dried diffusively as sessile droplets or desiccated under vacuum after they are adsorbed onto glass microfiber filters. The glass transition temperatures (Tg) of the desiccated samples were measured by temperature-ramp Fourier Transform Infrared (FTIR) spectroscopy. Sera samples vitrified at 4±2°C when 0.8M trehalose and 0.01M dextran were added and the samples were vacuum dried for two hours. Western immunoblotting showed that vitrified serum proteins were minimally degraded when stored for up to one month at 4°C. About 80% of all proteins were recovered after storage at 4°C on glass microfiber filters, and recovery did not decrease with storage time. These results demonstrated the feasibility of long-term storage of vitrified serum at hypothermic (and non-cryogenic) temperatures.
全世界的生物库都收集人类血清样本，并将其储存起来供未来研究使用。目前，全世界有数百个生物库将人类血清样本储存在冰箱、冰柜或液氮中，而没有遵循任何特定的冷冻保存方案。这种储存方法既昂贵又可能对生物样本造成损害。为了降低储存成本和冻融应激，我们探索了使用等温热玻璃化法在非低温条件下储存存档人类血清样本的可行性。当生物样本被玻璃化时，可以停止生化反应，样本停止降解，并且可以在不要求低温储存的情况下稳定大分子。在这项研究中，向人类血清样本中添加了 0.2、0.4 或 0.8M 海藻糖；0、0.005 或 0.01M 葡聚糖；以及 0 或 10%（v/v）甘油。这些样本要么以贴壁液滴的形式扩散干燥，要么在吸附到玻璃微纤维滤器后在真空中干燥。通过温度斜坡傅里叶变换红外（FTIR）光谱法测量干燥样品的玻璃化转变温度（Tg）。当添加 0.8M 海藻糖和 0.01M 葡聚糖并将样品真空干燥两小时时，血清样品在 4±2°C 下玻璃化。Western 免疫印迹显示，在 4°C 下储存长达一个月时，玻璃化血清蛋白的降解最小。在玻璃微纤维滤器上储存 4°C 时，约 80%的蛋白质被回收，并且随着储存时间的延长，回收率没有下降。这些结果表明，在低温（非低温）条件下长期储存玻璃化血清是可行的。
