Mazur Peter, Seki Shinsuke, Pinn Irina L, Kleinhans F W, Edashige Keisuke
Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932-2575, USA.
Cryobiology. 2005 Aug;51(1):29-53. doi: 10.1016/j.cryobiol.2005.04.008.
The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF.
冷冻过程中细胞内冰形成（IIF）的发生与否是决定细胞能否在冷冻保存中存活的唯一最重要因素。IIF的一个重要决定因素是过冷细胞发生核化的温度。为避免细胞内冰形成，细胞必须冷却得足够慢，以便在达到该温度之前，渗透脱水消除几乎所有的细胞过冷现象。本报告关注的是决定小鼠卵母细胞核化温度的因素。其中最主要的是冷冻保护添加剂（此处为甘油或乙二醇）的浓度。IIF的温度从缓冲等渗盐水（PBS）中的-14℃降至1M甘油/PBS和1.5M乙二醇/PBS中的-41℃。后者能迅速渗透到卵母细胞中；前者则不能。根据CPA浓度，在-3.9至-7.8℃开始的细胞外初始冷冻会使细胞变形。在PBS中，这种变形常常导致IIF；在CPA中则不会。卵母细胞被透明带包围。该结构似乎会阻碍外部冰穿过它生长，但不会阻止。在大多数情况下，IIF的特征是在冷却过程中突然变黑或闪烁。但在某些情况下，尤其是对于去透明带的卵母细胞，在冷却过程中会突然形成浅棕色的面纱，随后在升温过程中变黑速度较慢。在-30℃以上，闪烁在几分之一秒内发生。在-30℃以下，通常发生得要慢得多。我们观察到闪烁有时会伴随着细胞质的突然喷出。在冷冻过程中，细胞位于生长的外部冰之间未冻结的通道中。根据相图数据，我们计算了在观察到的闪烁温度下未冻结的水和溶液的比例以及这些通道中盐和CPA的浓度。关于这些特征中哪一个与IIF最相关，结果有些模糊。
