Zhang Miao, Oldenhof Harriëtte, Sieme Harald, Wolkers Willem F
Institute of Multiphase Processes, Leibniz Universität Hannover, Germany.
Clinic for Horses, Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany.
Biochim Biophys Acta. 2016 Jun;1858(6):1400-9. doi: 10.1016/j.bbamem.2016.03.020. Epub 2016 Mar 18.
The aim of this study was to investigate if membrane-impermeable molecules are taken up by fibroblasts when exposing the cells to membrane phase transitions and/or freezing-induced osmotic forces. The membrane-impermeable fluorescent dye lucifer yellow (LY) was used to visualize and quantify uptake during endocytosis, and after freezing-thawing. In addition, trehalose uptake after freezing and thawing was studied. Fourier transform infrared spectroscopic studies showed that fibroblasts display a minor non-cooperative phase transition during cooling at suprazero temperatures, whereas cells display strong highly cooperative fluid-to-gel membrane phase transitions during freezing, both in the absence and presence of protectants. Cells do not show uptake of LY upon passing the suprazero membrane phase transition at 30-10°C, whereas after freezing and thawing cells show intracellular LY equally distributed within the cell. Both, LY and trehalose are taken up by fibroblasts after freezing and thawing with loading efficiencies approaching 50%. When using 250 mM extracellular trehalose during cryopreservation, intracellular concentrations greater than 100 mM were determined after thawing. A plot of cryosurvival versus the cooling rate showed a narrow inverted-'U'-shaped curve with an optimal cooling rate of 40°C min(-1). Diluting cells cryopreserved with trehalose in isotonic cell culture medium resulted in a loss of cell viability, which was attributed to intracellular trehalose causing an osmotic imbalance. Taken together, mammalian cells can be loaded with membrane-impermeable compounds, including the protective agent trehalose, by subjecting the cells to freezing-induced osmotic stress.
本研究的目的是调查当将成纤维细胞暴露于膜相变和/或冷冻诱导的渗透力时，膜不可渗透分子是否会被成纤维细胞摄取。使用膜不可渗透的荧光染料路西法黄（LY）来可视化和量化内吞过程中以及冻融后的摄取情况。此外，还研究了冻融后海藻糖的摄取情况。傅里叶变换红外光谱研究表明，成纤维细胞在高于零摄氏度的温度冷却过程中显示出轻微的非协同相变，而在冷冻过程中，无论有无保护剂，细胞都会显示出强烈的高度协同的流体到凝胶的膜相变。细胞在30-10°C通过高于零的膜相变时不会摄取LY，而在冻融后，细胞内的LY在细胞内均匀分布。冻融后，LY和海藻糖均被成纤维细胞摄取，加载效率接近50%。在冷冻保存期间使用250 mM细胞外海藻糖时，解冻后细胞内浓度大于100 mM。冷冻存活率与冷却速率的关系图显示出一条狭窄的倒“U”形曲线，最佳冷却速率为40°C min(-1)。用海藻糖冷冻保存的细胞在等渗细胞培养基中稀释会导致细胞活力丧失，这归因于细胞内海藻糖导致渗透失衡。综上所述，通过使细胞承受冷冻诱导的渗透应激，哺乳动物细胞可以加载膜不可渗透的化合物，包括保护剂海藻糖。
