Institute of Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany.
Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine Hannover, Hannover, Germany.
Biochim Biophys Acta Biomembr. 2018 Feb;1860(2):467-474. doi: 10.1016/j.bbamem.2017.10.031. Epub 2017 Oct 31.
Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6M concentrations compared to only 1M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.
在细胞的冷冻保存或玻璃化过程中，膜是冷冻损伤的主要部位。添加冷冻保护剂（CPA）可以减少冷冻损伤，但也会干扰膜的完整性，导致细胞内成分泄漏。本研究的目的是在脂质体模型系统中研究脂质-CPA 相互作用，以深入了解冷冻保存或玻璃化处理过程中细胞保护和毒性的机制。研究了各种 CPA，包括二甲亚砜（DMSO）、甘油（GLY）、乙二醇（EG）、二甲基甲酰胺（DMF）和丙二醇（PG）。研究了在添加 CPA 以及冷冻和解冻后，对包封羧基荧光素（CF）的磷脂囊泡的磷脂囊泡泄漏的保护作用。使用傅里叶变换红外光谱研究了 CPA 与磷脂酰基链和头部基团的分子相互作用以及膜相行为。与测试的其他 CPA 相比，DMSO 对 PC-脂质体的影响明显不同，CF 保留和膜相行为的测量均如此。所有的 CPA 都被发现能在冷冻过程中抑制膜的通透性。然而，在冷冻之前，高浓度的 CPA 就会导致泄漏，其顺序为 DMSO、EG、DMF/PG 和 GLY。与 GLY 相比，DMSO 使脂质体能够承受高达 6M 的浓度。胆固醇添加到 PC-脂质体中增加了膜对通透性的稳定性。发现 DMSO 使磷脂头部基团脱水并提高膜相变温度，而其他 CPA 则使脂质头部基团的水合水平增加，同时降低膜相变温度。
