Measurement of the size of intracellular ice crystals in mouse oocytes using a melting point depression method and the influence of intracellular solute concentrations.

Characterization of intracellular ice formed during the cooling procedures of cells significantly benefits the development and optimization design of cryopreservation or cryosurgery techniques. In this study, we investigated the influence of the concentration of extracellular non-permeable and permeable solutes on the melting points of the intracellular ice in mouse oocytes using cryomicroscopy. The results showed that the melting points of the intracellular ice are always lower than the extracellular ice. Based on this observation and the Gibbs-Thomson relation, we established a physical model to calculate the size of intracellular ice crystals and described its relationship with the concentrations of intracellular permeating solutes and macromolecules. This model predicts that the increased concentration of macromolecules in cells, by increasing the extracellular non-permeating solute concentration, can significantly lower the required concentration of permeable solutes for intracellular vitrification. The prediction was tested through the cryomicroscopic observation of the co-existence of intracellular vitrification and extracellular crystallization during cooling at 100 degrees C/min when the extracellular solutions contain 5 molal (m) ethylene glycol and 0.3 to 0.6m NaCl.