Selecting a Cryoprotectant for Eukaryotic & Bacterial Cell Storage

The cryoprotectant your lab selects for long-term storage of both eukaryotic and bacterial cell storage can have profound implications for future experimental results. Cryoprotectants, literally “to protect cells and tissue from damage due to freezing,” can be man-made, or generated in nature. For example, some species of plants and animals produce substances with anti-freeze properties to help them survive cold environments. The sugar trehalose is one such compound, and is being tested as a cryoprotectant option for lab use.

这些细胞监护人通常用其他物质配方，例如蒸馏水，乙二醇和/或牛血清，以创建冻结的理想食谱。冷冻保护剂分为两类：细胞外和细胞内。细胞外的冷冻保护剂是非渗透分子，通过减少发生的过度细胞裂解作用
during the freezing process. Intracellular cryoprotectants penetrate the cell membrane and prevent the formation of ice crystals, which rupture cell membranes. Here we discuss the implications of using these solutions in cryopreservation of nucleus/organelle-containing eukaryotic cells, and more simply structured bacterial cells.

细胞外冷冻保护剂

细胞外冷冻保护剂不会穿透真核细胞或细菌细胞壁的膜。因此，它们无法防止由于细胞内水晶形成而导致的细胞膜破裂。这些非穿透分子​​的例子是蔗糖，葡萄糖和聚乙烯基吡咯烷酮（PVP）。

取而代之的是，这些分子的功能可减少作为样品通过冰冻过程过渡的过度渗透梯度。由于较少的水可以用作细胞外的溶质，因此盐浓度大大增加。细胞上渗透压的增加导致细胞形状，细胞相互作用，分子的亚细胞分布以及最终的细胞活力。

细胞外冷冻保护剂的存在可用于平衡渗透压差异并防止细胞中的水排出。而且，由于这些分子没有穿透膜，因此它们对细胞生理没有直接的，有害的影响。

Intracellular Cryoprotectants

Intracellular cryoprotectants, such as dimethyl sulfoxide (DMSO) and glycerol, have become mainstay reagents for cryopreservation. Both of these molecules have the ability to penetrate the cell membrane to reduce the formation of water crystals within the cell during the freezing process. To maintain cell viability when using these reagents, the freezing protocol must be slow and controlled, allowing the efflux of water to occur.

如果过程太快，则在水退出细胞之前会形成细胞内的冰晶体。

While DMSO and glycerol have been traditionally used for the cryopreservation of eukaryotic and bacterial cells respectively, there are still disadvantages to this approach. In some instances these two molecules have inverse effects on the same protein. In addition, different cell types have different permeability for these molecules. This difference affects the rate at which a cell shrinks and swells before freezing, during freezing, and during thaw cycles. This can have a dramatic impact on cell viability.

因此，需要对使用的冷冻保护剂的类型和浓度进行密切考虑。仔细分析有关细胞类型和小规模实验的标准操作程序的文献分析可以节省由于不良的冷冻保存条件而不得不重复漫长的实验的挫败感和成本。

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