Interfacial Interactions of Sucrose during Cryopreservation Detected by Raman Spectroscopy

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.