低温保存实验室样品，样本和试剂在生物医学研究实验室中会经常用到。本短片将讨论让实验室样品保持冷却的一些方法，还将阐明针对每个实验要求选择使用正确的冷却方法。例如，冰和干冰这一类冷却试剂，通常用来在实验过程中保持样品冷却。本视频讨论了最常用的冷却试剂的物理属性，以及操作使用它们时要注意的安全事项。若要在不同实验之间保存样品冷却，则需用到制冷仪器，包括实验室级别的冰箱和冻箱来对样品做长时间的保存。本视频还讨论了可用目前常用实验室制冷仪器存储的样品和试剂的类型。最后还介绍了超低温保存的概念，它是将组织，细胞和生物分子冷却到0度以下的过程，可有效终止所有降解样品的生物学反应。这里讨论了几种可以尽量减小或避免形成能造成损伤的冰晶的超低温保存方法。
冷却试剂的使用
对冷却试剂的选择取决于实验操作过程的性质。传统的冰块是短时间保存样品的合理选择。您应当知道冰是冻的水，如您在这个相图中看到的，在正常大气压下它的融化温度是0度。但您可能不知道它有时被称之为“湿冰”，因为在室温下它会受热而变成液体。“湿冰”是操作和转移样品以及试剂时的最佳选择。“湿冰”是纯的水，而“干冰”是固体形式的二氧化碳，它的融化温度是-78.5度。正常大气压下，干冰并不融化成液体，而是通过一种叫升华的过程直接转变成气体的二氧化碳。升华是指由固体直接转变成气体的形态改变，它发生在相图中的三相临界点之下。当您操作生物样本如冷冻的细菌，哺乳细胞或组织时，要使用干冰，这些样品通常都被保存在0度以下温度。干冰的还一个好处是它在形态改变时不会留下任何残余物，将液体浇在干冰上，使得它成为作冷冻浴池的最佳选择。
液氮是浓缩的氮气，通常用LN2来表示。在它的相图中您可以看到，正常大气压-196度时，液氮会沸腾，由液体变成气体。当您需要将生物样品保存在低于大多数实验室冻箱所能达到的温度是，就需要用到液氮。液氮可以存储在杜瓦瓶，一种真空瓶中，加上一个非密封的盖子，也可存储在一个大的杜瓦罐中，罐上装有排气阀门防止气压在罐内的累积增高。干冰和液氮虽然无毒害，但它们都是危险物品，您需要经过实验室有经验的人员训练指导后才能操作它们。由于液氮和干冰的极低温度，如果接触到皮肤都会造成严重的组织损伤。所以要时刻穿戴防护用具，包括耐低温手套和实验服。并使用工具来操作样品，避免直接接触到皮肤。此外，千万不要用密封的容器来储存干冰或者液氮，因为这些制冷物质会转变成气体。在密封条件下，累积起来的压力会导致爆炸。
制冷仪器的使用
现在来看看将样品保持冷却的仪器…实验室的冰箱和冻箱比我们家用的对温度控制要更加精确，来确保整个仪器内部有恒定的温度。它们通常都装有温度监控系统和报警器，当温度有显著变化时会发出警报。任何时候都不要在实验室的冰箱或冻箱中储存食物或饮料，因为会引起有害化学物或细菌污染。您需要用其他的地方来放置您的午饭。
冰箱温度保持在4度，通常用来短期存储样品，尤其是当冷冻会影响到样品完整性时。许多试剂和溶液存储在4度来延长它们的有效期，包括组织培养液，铺板好的细胞培养平板，使用前需要将它们预热。冷藏室最适合用来放置需要低温下操作的较大仪器，如液相色谱分析装置。
实验室级别的冻箱温度范围是-20度到-196度的低温冷冻箱。保存核酸和类似限制性酶一类的试剂，适合用-20 °C温度。样品和试剂从冻箱中取出后，需放置在冰上。在液氮中超低温保存的冷冻的组织和细胞，可放置在-80°C和低温冷冻箱继续保存一段时间。干冰通常是当样品从-80 ˚C冻箱取出时转移样品时用到。
应用
超低温保存是指对组织甚至活细胞进行长时间的存储。在0度以下时，所有的生物活动，包括降解样品的反应都被有效终止了。冷冻活细胞和组织时会形成冰晶造成细胞脱水和损伤，还会导致溶质分子积累到有毒害的高浓度。急冻或称速冻是将生物样品迅速浸没在液氮中或干冰与乙醇混合物中的过程，这样不会形成大的冰晶而损坏细胞。冷冻保护剂液也可用作添加物来减少冰晶的形成。
除了速冻法，还可以用机器来缓慢调节冷冻过程，如您在这里看到的低温冷冻羊胚胎所需要的过程。最近又新出了玻璃化冷冻法，它可超低温冷冻细胞和组织而不会有任何冰晶造成的损伤。这一过程利用某些冷冻保护剂进行快速冷冻，将样品中的液体转变成一种非晶体状，玻璃化的固体。
结语
您刚观看的是JoVE对冷却实验室样本和试剂的介绍。本短片中，我们回顾了冷冻试剂和仪器的不同类型，以及何时使用每种冷却方法的例子。我们还介绍了超低温保存生物学样本的几种方法。感谢观看。
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