PDF(Pubmed)
Abstract：
Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.
摘要：
冰的损害和抑冰冷冻保护剂（CPAs）的潜在毒性是人类辅助生殖的关键问题，家畜和研究动物，和使用冷冻保存的卵母细胞和胚胎的濒危物种。使用基于同步加速器的时间分辨X射线衍射检查了快速冷却和快速升温过程中牛卵母细胞（大小与人类和大多数其他哺乳动物的卵母细胞相似）中形成的冰的性质。使用冷却速率,当前实践的增温率和CPA浓度，卵母细胞在冷却后没有显示出冰，但在升温过程中总是产生大的冰部分-与大多数游离水的结晶一致，所以大多数与冰有关的损害必须在变暖期间发生。加温时冰的详细行为取决于冷却过程中形成的冰的性质。增加冷却速率允许在当前实践中浸泡的卵母细胞在冷却和加温期间保持基本上无冰。证明了更大的对流升温速率，这将允许常规的无冰冷冻保存，CPA浓度较小。这些结果阐明了冷却的作用，变暖,和CPA浓度在卵母细胞中生成冰，并建立形成的冰的结构和粒度。在许多物种中，冰的形成可以作为影响增温后卵母细胞活力和发育的因素被消除。改善结果，并允许对冷冻保存周期的其他有害影响进行独立研究。
