Vajta G, Holm P, Kuwayama M, Booth P J, Jacobsen H, Greve T, Callesen H
Embryo Technology Center, Danish Institute of Agricultural Sciences, Tjele.
Mol Reprod Dev. 1998 Sep;51(1):53-8. doi: 10.1002/(SICI)1098-2795(199809)51:1<53::AID-MRD6>3.0.CO;2-V.
Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000 degrees C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over -180 degrees C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction.
尽管目前对某些哺乳动物胚胎进行冷冻保存已是常规操作，但根据胚胎阶段、物种及来源（体内或体外产生）的不同，冷冻效率仍存在显著差异。据推测，造成这些差异的主要因素包括细胞内脂滴的数量、导致冷损伤的不同微管结构，以及影响冷冻保护剂渗透的体积/表面积比。一种新方法——开放式拉长细管（OPS）法，其冷却和升温速率极高（超过20,000℃/分钟），且与浓缩冷冻保护添加剂的接触时间很短（在-180℃以下少于30秒），为避免冷损伤以及减少毒性和渗透损伤提供了可能。在本文中，我们报告了采用OPS法对不同发育阶段的体外生产牛胚胎进行玻璃化冷冻的情况。从第3天到第7天（第0天 = 受精日）冷冻保存的胚胎发育成囊胚的比率与对照胚胎相当；即使是在第1天或第2天冷冻保存的胚胎，其存活率也只是略有降低。81%的第8天孵化囊胚也在该操作后存活下来。该方法也成功应用于牛卵母细胞；184个玻璃化冷冻的卵母细胞中，25%在受精并培养7天后发育成囊胚。在卵母细胞和囊胚阶段进行玻璃化冷冻后移植均成功受孕。OPS玻璃化冷冻为解决生殖低温生物学的基本问题提供了一种新途径，可能对动物生物技术和人类辅助生殖产生实际影响。
