Cryopreservation: Vitrification and Controlled Rate Cooling
Cryopreservation: Vitrification and Controlled Rate Cooling
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DOI：
10.1007/978-1-4939-6921-0_5
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Hunt, Charles J.
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作者：
Hunt, Charles J.
低温保存是应用低温来保存细胞和组织的结构和功能的完整性。在低温保存过程中，传统的冷却方案允许冰的形成和溶质浓度的上升。溶质浓度升高所造成的损害可以通过使用称为冷冻保护剂的化合物来减轻。这些化合物保护细胞免受缓慢冷却损伤的后果，使它们以冷却速度冷却，从而避免细胞内冰的致命影响。一种替代传统冷却的方法是玻璃化。玻璃化方法在足够高的浓度下加入冷冻保护剂，以防止冰结晶，使系统形成非晶玻璃，从而避免由传统的缓慢冷却引起的破坏性影响。然而，玻璃化也会对细胞造成破坏性后果，因为在较低的冷却速率下玻璃化细胞所需的冷冻保护剂浓度是潜在的，而且往往是有害的。虽然这些浓度可以降低到无毒水平，但如果细胞被超快速冷却，如果处理不当，在随后的储存和再加热过程中，由此产生的亚稳态系统可能会通过脱硝和冰的生长而导致损伤。干细胞的商业和临床应用需要稳健且可重复的低温保存方案和适当的长期低温储存条件，以提供可靠的主细胞库和工作细胞库。尽管目前符合《药品生产质量管理规范》（cGMP）的临床级多能干细胞的衍生和储存方法已经存在，而且也有适合临床应用的干细胞系，但目前的冷冻保存方案，无论是玻璃化冷冻还是传统的慢速冷冻，仍然不是最理想的。除了因次优冷冻保存而导致的有价值产品的损失外，还有一种危险，即这种过程将对细胞施加选择性压力，选择出不具代表性的抗冷冻亚群。优化这一过程需要了解细胞系统冻结过程中发生的基本过程，损伤机制和避免它们的方法。本章汇集了在其他系统中获得的冷冻保存知识，以及当前胚胎和诱导多能干细胞保存的最新技术，试图为未来优化冷冻保存方案的尝试提供背景。
Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cooling injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at sufficiently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and growth of ice during subsequent storage and rewarming if not appropriately handled.The commercial and clinical application of stem cells requires robust and reproducible cryopreservation protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical applications are available, current cryopreservation protocols, whether for vitrification or conventional slow freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreservation engenders, there is a danger that such processes will impose a selective pressure on the cells selecting out a nonrepresentative, freeze-resistant subpopulation. Optimizing this process requires knowledge of the fundamental processes that occur during the freezing of cellular systems, the mechanisms of damage and methods for avoiding them. This chapter draws together the knowledge of cryopreservation gained in other systems with the current state-of-the-art for embryonic and induced pluripotent stem cell preservation in an attempt to provide the background for future attempts to optimize cryopreservation protocols.