Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification

The failure of complex mammalian organs, such as the kidney, to function following freezing to low temperatures is thought to be due largely to mechanical disruption of the intercellular architecture by the formation of extracellular ice. Classical approaches to the avoidance of ice formation through the imposition of ultra-rapid cooling and warming rates or by gradual depression of the equilibrium freezing point during cooling to -80 degrees C have not been adequate. An alternative approach relies on the ability of highly concentrated aqueous solutions of cryoprotective agents to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify without the formation of ice, a process termed vitrification. When embryo suspensions are cryopreserved using conventional procedures, this supercooling behaviour allows intracellular vitrification, even in the presence of extracellular ice. We have therefore used mouse embryos to examine the feasibility of obtaining high survival following vitrification of both the intra- and extracellular solutions and report here that in properly controlled conditions embryos seem to survive in high proportions after cryopreservation in the absence of ice.     

Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo

The widespread use of clomiphene citrate and exogenous gonadotrophins for in vitro fertilization (IVF) in human frequently results in the production of multiple embryos. Replacement of more than two embryos increases pregnancy rate but may result in multiple pregnancies with increased pre- and post-natal abnormality. Preservation of embryos for a limited time allows fewer embryos to be replaced on several different occasions and thus the problems of multiple pregnancy can be minimized, the effectiveness of a single IVF procedure increased and embryo replacement in adverse maternal conditions avoided. Preimplantation embryos have been successfully cryopreserved in many animal species. The sensitivity of embryos to cooling and freezing varies between species and stages of embryo development. We report here the cryopreservation procedures that allow a high survival rate of four- and eight-cell human embryos and the establishment of a pregnancy following the freezing and storage of an eight-cell embryo for 4 months in liquid nitrogen. The pregnancy terminated at 24 weeks' gestation due to development of a septic Streptomyces agalactiae chorion amnionitis after premature membrane rupture.     

Cryogenic Thermophysical Studies for Clinical Medicine

1　Introduction of Cell Injury DuringFreezing and ThawingAs biological cells and tissues contain much water,they can be analyzed as aqueous systems.Twosub- processes occur during freezing:heat transferand mass transfer.Heat is removed to thesurroundings s…     

乙二醇玻璃化冷冻液二步法对长爪沙鼠 2-细胞胚胎的冷冻保存

摘要: 目的 评价用乙二醇玻璃化冷冻液二步法对长爪沙鼠 2-细胞胚胎冷冻保存的效果。方法 通过超排卵处理 后与雄鼠交配获得长爪沙鼠 2-细胞胚胎,胚胎在冷冻液 EFS20 中平衡 3 min,然后移入冷冻液 EFS40 中平衡 1 min, 之后将细胞冻存管直接浸入液氮中保存。采用二步法复苏胚胎,复苏后移入改良的胚胎培养液( mKSOM) 中体外 培养。结果 采用该方法冷冻的胚胎复苏后胚胎回收率为 86. 73% ,存活率为 82. 94% ,复苏后存活的胚胎中约 1 / 3 胚胎可以在体外发育到囊胚。结论 乙二醇玻璃化冷冻液适于冷冻保存长爪沙鼠 2-细胞胚胎。     

超声辅助冷冻对湿面筋蛋白中可冻结水含量及冰晶粒度分布的影响

超声辅助食品冷冻过程是一种新的技术。本文通过对湿面筋蛋白中可冻结水含量及冰晶粒度分布的检测,研究超声辅助食品冷冻过程中冰结晶的热力学机制。用差示扫描量热仪测定了不同冷冻环境下湿面筋蛋白中可冻结水的含量,结果可见：超声辅助冷冻和液氮浸渍冷冻的湿面筋蛋白中可冻结水的含量比传统冷冻过程的高。用扫描电镜间接观察和分析了冷冻的湿面筋蛋白中冰晶的形状和粒度分布,表明适当的功率超声能加速湿面筋蛋白的冷冻过程;超声辅助和液氮浸渍冷冻的湿面筋蛋白中冰晶细小且分布均匀,可减少冷冻时对面筋蛋白网络结构的破坏。显示超声辅助冷冻是改善冷冻食品品质的实用技术。     

小样品在过冷液氮中淬冷时冷却速率的实验研究

本文通过小样品在常压过冷液中的淬冷实验，揭示了液氮过冷度和样品尺寸对小样品淬冷时冷却速率的影响．实验结果表明，增加液氮过冷度、减小样品尺寸均能有效地提高样品的冷却速率，对小生物体的低温保存和低温固定非常有利．     

从蔷薇科果树硅胶干燥叶片中制备DNA

以6种蔷薇科主要树种特别是杏属植物为试材，从DNA提取、模板DNA电泳分析、RAPD扩增和S-基因型特异PCR4个方面对硅胶干燥的样品与传统液氮冷冻的样品进行了综合对比研究，结果表明：硅胶保存的样品完全可以与传统液氮冷冻的样品得到同样的PCR扩增结果，甚至在减小褐变影响方面优于后者，且干制样品的DNA纯度均比液氮速冻样品高。研究表明，用硅胶干燥法保存的植物样品可以满足后期有关RAPD等分子生物学研究的需要，且能保证野外考察时植物样品的DNA在运输过程中不被降解。     

Structural transformation in supercooled water controls the crystallization rate of ice

One of water's unsolved puzzles is the question of what determines the lowest temperature to which it can be cooled before freezing to ice. The supercooled liquid has been probed experimentally to near the homogeneous nucleation temperature, T(H) ≈ 232 K, yet the mechanism of ice crystallization-including the size and structure of critical nuclei-has not yet been resolved. The heat capacity and compressibility of liquid water anomalously increase on moving into the supercooled region, according to power laws that would diverge (that is, approach infinity) at ~225 K (refs 1, 2), so there may be a link between water's thermodynamic anomalies and the crystallization rate of ice. But probing this link is challenging because fast crystallization prevents experimental studies of the liquid below T(H). And although atomistic studies have captured water crystallization, high computational costs have so far prevented an assessment of the rates and mechanism involved. Here we report coarse-grained molecular simulations with the mW water model in the supercooled regime around T(H) which reveal that a sharp increase in the fraction of four-coordinated molecules in supercooled liquid water explains its anomalous thermodynamics and also controls the rate and mechanisms of ice formation. The results of the simulations and classical nucleation theory using experimental data suggest that the crystallization rate of water reaches a maximum around 225 K, below which ice nuclei form faster than liquid water can equilibrate. This implies a lower limit of metastability of liquid water just below T(H) and well above its glass transition temperature, 136 K. By establishing a relationship between the structural transformation in liquid water and its anomalous thermodynamics and crystallization rate, our findings also provide mechanistic insight into the observed dependence of homogeneous ice nucleation rates on the thermodynamics of water.     

DAP 玻璃化冷冻液对小鼠2-细胞胚胎的冷冻保存

摘要: 目的探索和优化小鼠2-细胞胚胎冷冻效果,建立小鼠胚胎冷冻保存技术以及超低温保存模型动物胚胎实验室所需的相关技术。方法选择3 个品系( C57BL/6、ApoE － / －、NOS3 － / － ) SPF 级4 周龄雌性小鼠,运用超数排卵、体外受精、玻璃化冷冻法,以及二细胞期胚胎复苏、体外培养等技术方法,观察上述实验效果。结果C57BL/6、ApoE － / －、NOS3 － / － 3 个品系小鼠超数排卵平均值分别为42. 98 个/只、27. 93 个/只、23. 11 个/只,体外受精2-细胞期胚胎率分别为68. 79%、32. 70%、23. 08 %,胚胎冷冻复苏后胚胎回收率72. 77%、80. 87%、83. 33%,存活率分别为56. 92%、54. 84%、70%,存活胚胎体外培养发育到囊胚比率分别为72. 37%、60. 78%、25%。结论选择4 周龄小鼠,按预定相同的技术方法做超数排卵、体外受精、胚胎冻存、胚胎复苏,呈现出较好效果,C57BL/6 小鼠优于基因工程小鼠,基本建立起小鼠2-细胞胚胎冷冻保存技术。     

低温在医学上的应用

介绍了低温在医学上的应用，它包括冷冻外科、低温保存、低度体温、低温干燥4个方面，冷冻医疗主要用液氮作低温制剂，它价廉、简便、易行。已广泛应用于临床各科治疗良性及恶性肿瘤，疗效确切，另在低温保存血液、精液、组织、器官方面成绩可，有待于进一步研究发展。     

液氮冷却作用下三类高温岩石力学性能试验研究

为了研究液氮冷却对高温岩石物理力学性能的影响，对不同温度下(25~350℃)的花岗岩、片麻岩和砂岩试样进行液氮冷却处理，开展了一系列的物理力学试验研究，结合微观观察结果分析了各类岩石的损伤机理.结果表明，提高加热温度能够加剧液氮对岩石内部结构的损伤，随温度的升高，岩石的孔隙率、峰值应变逐渐增大，而纵波波速、抗压强度和弹性模量则相反.高温和液氮冷却所产生的热应力导致岩石内部裂纹的萌生和扩展，且微裂纹主要沿石英矿物边界发育.随着加热温度的升高，微裂纹的数量呈逐渐增加的趋势，这是岩石宏观特性退化的主要原因.三类岩石对加热和液氮冷却处理的敏感度不同，这与岩石在成岩作用、矿物成分、胶结类型以及孔隙结构方面的差异有关.     

几种林果低温贮藏的冷害及其调控研究进展

冷害可造成林果采后低温贮运损失．使其食用品质下降，甚至不能食用。笔综述了林果的冷害症状、冷害机理，并重点介绍了如何防治林果低温贮藏冷害的方法，其中包括变温贮藏、化学物质处理、气调贮藏及其他方法。最后探讨了这些方法在生产中应用的可能性及存在的问题，提出了冷害修复可作为林果冷害防治进一步研究的方向。     

全球变暖会导致气候严寒吗

文章对美国国防部“秘密报告”中全球变暖可能导致严寒的理论根据和现实可能性作了分析，指出由于气候变暖、极冰融化影响大洋的环流，进而影响全球气候；这样的事件在历史上曾有多次发生。但目前还不能说已经开始发生这样的过程。而且没有这样的先兆。因此，到2020年，发生“秘密报告”中所提出来的情景的可能性很小，但是不排除在更长时间发生某种气候受变的可能性，所以加强对气候突变的研究是非常必要的。     

Au-Cu-Al合金中的马氏体转变

采用3种热处理工艺研究了Au-Cu-Al合金母相的有序化程度,分析了其A2→B2→L21的有序化转变对马氏体转变的影响,即其相变点、马氏体和母相的结构,以及400℃时效炉冷处理后没有生成马氏体的原因.结果表明,采用100℃时效1.5 h后淬火到冰水中(热处理工艺II)的热处理工艺,可以得到表面浮突的马氏体;采用从650℃直接淬火到液氮中(热处理工艺I)的热处理工艺,也会发生马氏体转变,且其均为bct结构,时效处理可使母相得到充分有序化,从而提高其马氏体转变点温度,并使马氏体转变更加充分;此外,采用400℃时效、炉冷处理并淬火(热处理工艺III)的热处理工艺,可以获得更高的母相有序度,且不会导致马氏体相变,其所产生的bct结构的新相可能是通过形核长大转变而来.     

公鸭、公鸡精子对在液氮中低温冷冻保存耐受力的比较研究

比较公鸭和公鸡精子经低温冷冻保存后存活率、顶体完整率方面的差异.结果表明,公鸭冷冻精液解冻后的精子存活率和顶体完整率都不同程度地高于相同类型的公鸡冷冻精液,但差异均未达到显著水准（P＞0.05）.     

磁场影响水溶液冰晶的试验及装置

为寻找生物组织低温无损保存的新方法,研究了磁场对KMnO4溶液冰晶形成的影响.建立了不同形式的磁场发生装置,包括旋转磁场、方波叠加正弦波磁场、50kHz磁场.为保证样品降温过程慢速均匀,运用虚拟仪器技术建立了温度测控系统.使用低温显微系统对磁场影响下样品冰晶的形态进行了记录.结果表明,低频旋转磁场对生物溶液冰晶的形成产生了影响,尤其50kHz交变磁场对溶液中冰晶的生长具有较为显著的抑制作用,说明在易于产生的低冷冻速率下,交变磁场对生物组织的低温无损保存研究具有重要意义,适合实际应用.     

云南高原水稻品种干胚膜脂脂肪酸的不饱和度与抗冷性关系

对云南高原不同海拔、不同产地的十几种不同抗冷性的水稻品种进行了干胚膜脂的脂肪酸分析，研究表明水稻干胚膜脂脂肪酸的不饱和度高低与大田自然鉴定的抗冷性强弱有很强的正相关性，认为水稻干胚膜脂脂肪酸不饱和度高低确定可作为水稻开花结实期抗冷性鉴定的一个参考指标。     

具有液氮和蒸发氦气冷却屏的液氦容器的最佳热设计—能量优化法

本文提出了一种液氦容器最佳热设计的方法,它适用于利用液氮和蒸发的氦气冷却支撑和多层绝热的液氦容器。用以确定热交换器在支撑上和多层绝热间的最佳位置,可使液氮机和液氦机所消耗的总能量为最小。采用的优化方法是拉格朗日乘数法。并进行了归一化处理使得出的结果能适用于不同效率的液氮机和液氦机以及不同大小的杜瓦。对具有不锈钢支撑和环氧玻璃纤维支撑的液氦容器分别进行了计算,其计算结果用曲线表示,可便于设计者使用。