Langmuir. 2019 Jun 11;35(23):7388-7395. doi: 10.1021/acs.langmuir.8b01616. Epub 2018 Nov 14.
There is considerable interest in the use of sugars to preserve cells. In this study, low temperature Raman spectroscopy was used to characterize the behaviors of sucrose during freezing. The hydrogen bond network between sucrose and water was investigated at -10 °C and -50 °C, and the Raman spectra showed strengthened sucrose-water and sucrose-sucrose hydrogen bonds in more concentrated sucrose solution at -50 °C. The concentration of sucrose at the ice interface increased as the ice density decreased, and it plateaued across a narrow channel of nonfrozen sucrose solution before it decreased toward the next ice interface. The biophysical environment at interfaces between the cell and nonfrozen sucrose solution and between the cell and extracellular ice was also studied. A thin layer of nonfrozen sucrose solution was observed at the interface between the cell and extracellular ice. The extracellular concentration of sucrose at this interface was generally lower than that of bulk nonfrozen sucrose solution. The variation of sucrose concentration outside different regions of the cell membrane suggests that the chemical environment around the cell during freezing may be more heterogeneous than previously thought. Raman spectra and images also showed colocalization of nonfrozen sucrose solution and the cell, which implied that direct interaction between sucrose and cell membrane might be responsible for protective properties of sucrose. Sucrose was predominantly distributed outside the cell, and the observation of strong partitioning of sucrose across the cell membrane is consistent with substantial cell dehydration detected by the Raman spectra. This work enhances our understanding of the behaviors of sucrose solution and its interactions with cells at low temperature and can improve cryopreservation protocols of cells frozen in a sucrose-based media.
人们对使用糖来保存细胞非常感兴趣。在这项研究中，低温拉曼光谱被用于研究蔗糖在冷冻过程中的行为。在-10°C 和-50°C 下研究了蔗糖和水之间氢键网络，拉曼光谱显示，在-50°C 下更浓的蔗糖溶液中蔗糖-水和蔗糖-蔗糖氢键增强。冰界面处蔗糖的浓度随着冰密度的降低而增加，在靠近下一个冰界面之前，在非冻结蔗糖溶液的狭窄通道内达到一个平台，然后再降低。还研究了细胞与非冻结蔗糖溶液之间以及细胞与细胞外冰之间界面处的生物物理环境。在细胞和细胞外冰之间的界面处观察到一层薄薄的非冻结蔗糖溶液。该界面处细胞外蔗糖的浓度通常低于非冻结蔗糖溶液的整体浓度。细胞膜不同区域外蔗糖浓度的变化表明，冷冻过程中细胞周围的化学环境可能比以前想象的更加不均匀。拉曼光谱和图像还显示非冻结蔗糖溶液与细胞的共定位，这意味着蔗糖与细胞膜之间的直接相互作用可能是蔗糖具有保护性能的原因。蔗糖主要分布在细胞外，观察到蔗糖强烈地穿过细胞膜分配，这与拉曼光谱检测到的大量细胞脱水一致。这项工作增进了我们对低温下蔗糖溶液行为及其与细胞相互作用的理解，并可以改进基于蔗糖的细胞冷冻保存方案。
