Mazur P, Cole K W, Schreuders P D, Mahowald A P
University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences.
Cryobiology. 1993 Feb;30(1):45-73. doi: 10.1006/cryo.1993.1006.
Because of their high susceptibility to chilling injury, permeabilized Drosophila embryos can not be cryobiologically preserved by slow freezing at rates low enough to prevent the formation of intraembryonic ice. Calculations indicated that to outrun the chilling injury they must be cooled and warmed rapidly at an estimated 20,000 degrees C/min or faster. Ordinarily, such cooling rates would inevitably produce lethal intracellular ice. To prevent this, embryos must contain and be surrounded by sufficiently high concentrations of glass-promoting solutes to induce vitrification on cooling and prevent devitrification on warming. Like Steponkus et al. (Nature 345, 170, 1990) we have used ethylene glycol as the solute and have exposed permeabilized 12-h embryos to it in two steps. (Permeabilization was effected by exposing dechorionated embryos to a mixture of 0.3% 1-butanol in n-heptane for 90 or 110 s.) The two steps were (i) a 30-min exposure to 2 M ethylene glycol at 23 degrees C and (ii) a 5-min exposure to 8.5 M ethylene glycol [+/- 10% polyvinylpyrrolidone (PVP)] at 5 degrees C. The volumetric response to the first step indicates that full permeation of the 2 M glycol has been approached by 30 min. The point of the second step is to raise the intraembryonic concentration of ethylene glycol to near 8.5 M ethylene glycol by osmotic dehydration. Survival based on hatching is some 45% at this point. When 12-h embryos in 8.5 M glycol containing 10% PVP are then cooled to -205 degrees C at approximately 100,000 degrees C/min and warmed at about that rate, an average of about 12% survive (hatch), although in about half the runs 15-29% survive. Survivals in the absence of PVP are usually poorer but have been as high as 40%. Currently, 5% of the surviving larvae develop to adult flies (Steponkus et al. reported 18% hatching and 3% development to adult). Embryos that develop but do not hatch show readily detectable abnormalities in mouth parts and dorsal closure. Very high warming rates are much more critical to survival than are very high cooling rates; for example, none survive when warming is 2000 degrees C/min. The deleterious effect of slow warming is exerted between -80 and -40 degrees C. The lack of reciprocity between the effects of time spent cooling and time spent warming argues against ascribing death to chilling injury. Rather, it and other data argue for ascribing death to the devitrification during warming of cytoplasm that vitrified during cooling.(ABSTRACT TRUNCATED AT 250 WORDS)
由于果蝇胚胎对冷害高度敏感，通透化的果蝇胚胎无法通过以足够低的速率缓慢冷冻来进行低温生物学保存，以防止胚胎内结冰。计算表明，为了避免冷害，它们必须以估计每分钟20000摄氏度或更快的速度快速冷却和升温。通常情况下，这样的冷却速率会不可避免地产生致命的细胞内结冰。为了防止这种情况，胚胎必须含有并被足够高浓度的促进玻璃化的溶质包围，以便在冷却时诱导玻璃化并在升温时防止反玻璃化。和斯特庞库斯等人（《自然》345卷，第170页，1990年）一样，我们使用乙二醇作为溶质，并分两步将通透化的12小时胚胎暴露于其中。（通透化是通过将去壳胚胎暴露于正庚烷中0.3%的1-丁醇混合物中90或110秒来实现的。）这两步是：（i）在23摄氏度下将胚胎暴露于2M乙二醇中30分钟；（ii）在5摄氏度下将胚胎暴露于8.5M乙二醇[±10%聚乙烯吡咯烷酮（PVP）]中5分钟。对第一步的体积响应表明，到30分钟时2M乙二醇已接近完全渗透。第二步的目的是通过渗透脱水将胚胎内乙二醇的浓度提高到接近8.5M乙二醇。此时基于孵化的存活率约为45%。当含有10%PVP的8.5M乙二醇中的12小时胚胎随后以约每分钟100000摄氏度的速度冷却至-205摄氏度并以大致相同的速度升温时，平均约12%的胚胎存活（孵化），尽管在大约一半的实验中15%-29%的胚胎存活。在没有PVP的情况下存活率通常较低，但高达40%。目前，5%的存活幼虫发育成成年果蝇（斯特庞库斯等人报告孵化率为18%，发育成成年果蝇的比例为3%）。发育但未孵化的胚胎在口器和背闭合方面表现出易于检测到的异常。非常高的升温速率对存活比非常高冷却速率更为关键；例如，当升温速度为每分钟2000摄氏度时，没有胚胎存活。缓慢升温的有害影响在-80摄氏度至-40摄氏度之间显现。冷却时间和升温时间的影响缺乏对等性，这表明不能将死亡归因于冷害。相反，这以及其他数据表明，死亡是由于冷却时玻璃化的细胞质在升温过程中发生反玻璃化所致。（摘要截短至250字）
