NONEQUILIBRIUM FREEZING OF ONE-CELL MOUSE EMBRYOS - MEMBRANE INTEGRITY AND DEVELOPMENT POTENTIAL
NONEQUILIBRIUM FREEZING OF ONE-CELL MOUSE EMBRYOS - MEMBRANE INTEGRITY AND DEVELOPMENT POTENTIAL
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DOI：
10.1016/s0006-3495(93)81562-5
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发表时间：
1993-06-01
影响因子：
3.4
通讯作者：
ARMANT, DR
中科院分区：
文献类型：
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作者：
TONER, M;CRAVALHO, EG;ARMANT, DR
利用热力学模型对单细胞小鼠胚胎在不使用冷冻保护剂（CPAs）的情况下的快速三步非平衡冷冻方案进行了评价和优化，以避免致命的细胞内结冰（IIF）。在低温下使用低温显微镜观察单细胞小鼠胚胎的生物物理参数（即质膜的透水性，其温度依赖性和异质IIF参数）。然后将这些参数纳入热力学模型，该模型预测了IIF的可能性。模型预测表明，当在-10℃下插入5分钟的保温时间以确保细胞脱水时，在120℃/min的冷却速率下可以防止IIF的发生。该预测的冷冻方案在没有cpa的情况下避免了IIF，比传统的胚胎冷冻保存冷却速度（0.5至1℃/min）快两个数量级。在缓慢的冷却速率下，胚胎主要遵循平衡相图，不经历IIF，但IIF以外的机制（如高电解质浓度、机械效应等）会导致细胞损伤。我们使用可编程冷冻机测试了热力学模型的预测，并证实了理论预测。单细胞小鼠胚胎的膜完整性，通过双醋酸荧光素保留评估，通过我们的模型衍生的快速非平衡方案，在冷冻至-45℃后，膜完整性约为80%。事实上，胚胎可以在没有CPAs的情况下快速冷冻而不损害质膜，这是一个令人兴奋的新发现。为了保持胚胎的发育能力，有必要进一步完善这一方案。
A thermodynamic model was used to evaluate and optimize a rapid three-step rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120-degrees-C/min when a 5-min holding period was inserted at -10-degrees-C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1-degrees-C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other that IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45-degrees-C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.