Isothermal vitrification methodology development for non-cryogenic storage of archival human sera.
Isothermal vitrification methodology development for non-cryogenic storage of archival human sera.
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DOI：
10.1016/j.cryobiol.2013.01.003
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发表时间：
2013-04
期刊：
影响因子：
2.7
通讯作者：
Aksan, Alptekin
中科院分区：
文献类型：
--
作者：
Less, Rebekah;Boylan, Kristin L. M.;Skubitz, Amy P. N.;Aksan, Alptekin
关键词：
世界各地的生物储存库收集人类血清样本并储存它们以供未来研究。目前，世界上有数百个生物储存库将人类血清样品储存在冰箱、冷冻机或液氮中，而不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有害。为了降低储存成本和冻融应力，我们探索了使用等温玻璃化在非低温温度下储存存档人血清样品的可行性。当生物标本被玻璃化时，生化反应可以停止，标本停止降解，大分子可以稳定，而不需要低温储存。在本研究中，向人血清样品中加入0.2、0.4或0.8 M海藻糖; 0、0.005或0.01 M葡聚糖;和0或10%（v/v）甘油。将样品作为固着液滴扩散干燥或在它们吸附到玻璃微纤维过滤器上之后在真空下干燥。干燥样品的玻璃化转变温度（Tg）通过温度斜坡傅里叶变换红外（FTIR）光谱测量。当加入0.8 M海藻糖和0.01 M葡聚糖时，血清样品在4 ± 2 °C下玻璃化，并将样品真空干燥2小时。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程度最低。在4 °C下储存在玻璃微纤维过滤器上后，约80%的所有蛋白质被回收，并且回收率不随储存时间而降低。这些结果证明了玻璃化血清在低温（和非低温）下长期储存的可行性。
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 0.8 M trehalose; 0, 0.005 or 0.01 M 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 0.8 M trehalose and 0.01 M 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.
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