Liu J, Woods E J, Agca Y, Critser E S, Critser J K
Cryobiology Research Institute, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Biol Reprod. 2000 Nov;63(5):1303-12. doi: 10.1095/biolreprod63.5.1303.
Current mammalian embryo cryopreservation protocols typically employ an interrupted slow freezing (ISF) procedure. In general, ISF consists of initial slow cooling, which raises the extracellular solute concentration, and results in cell dehydration. Permeating cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are typically included in the medium to protect the cells against high solute concentrations. As this ISF procedure continues, slow cooling is terminated at an intermediate temperature (T(p)), followed by plunging into liquid nitrogen (LN(2)). If the slow cooling step allowed a critical concentration (CPA) of CPA to be reached within the cell, the CPA will interact with the remaining intracellular water during rapid cooling, resulting in the majority of the intracellular solution becoming vitrified and preventing damaging intracellular ice formation (IIF). This study presents a theoretical model to develop efficient ISF procedures, on the basis of previously developed data for the rat zygote. The model was used to select values of initial CPA concentrations and slow cooling rates (from initial estimated ranges of 0 to 4 molal DMSO and 0 to 2.5 degrees C/min cooling rates) that would allow the intracellular solute concentration to exceed the critical concentration. The optimal combination was then determined from this range based on minimizing the duration of slow cooling.
当前的哺乳动物胚胎冷冻保存方案通常采用间断慢速冷冻（ISF）程序。一般来说，ISF包括初始慢速冷却，这会提高细胞外溶质浓度并导致细胞脱水。渗透型冷冻保护剂（CPA），如二甲基亚砜（DMSO），通常包含在培养基中以保护细胞免受高溶质浓度的影响。随着这个ISF程序的继续，慢速冷却在中间温度（T(p)）终止，然后投入液氮（LN(2)）中。如果慢速冷却步骤能使细胞内达到CPA的临界浓度（CPA），那么在快速冷却过程中，CPA将与剩余的细胞内水分相互作用，导致大部分细胞内溶液玻璃化，并防止有害的细胞内冰晶形成（IIF）。本研究基于先前为大鼠受精卵开发的数据，提出了一个理论模型来制定高效的ISF程序。该模型用于从初始估计范围（0至4摩尔DMSO和0至2.5℃/分钟的冷却速率）中选择初始CPA浓度和慢速冷却速率的值，以使细胞内溶质浓度超过临界浓度。然后根据最小化慢速冷却持续时间从该范围内确定最佳组合。
