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

Abstract

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.

Fig. 1

A schematic of the cryomicroscopic observation using the sandwiched sample: A. Mouse oocytes are sandwiched into two quartz chips, whose distance is controlled by a two pieces of tapes of ~100μm thick; B. During freezing, both intracellular and extracellular solutions freeze; C. The melting of ice during warming.

Fig. 2

One example (0.15m NaCl and 3m EG as initial concentrations) of the significant difference between the melting behavior of intracellular and extracellular solutions: A. at −40°C during warming, cells and solutions are still frozen; B. at −9°C, cells become transparent but the extracellular solutions are still partially frozen; C. at −7°C, most of the extracellular ice melts.

Fig. 3

The comparison between the melting points of intracellular and extracellular solutions in the whole initial NaCl and EG concentration range: the upper curved plane is for the extracellular solutions and the lower one is for the intracellular solutions.

Fig. 4

The logarithm of the size of intracellular ice crystals (r) as a bi-linear function of extracellular NaCl and EG molality.

Fig. 5

A schematic of the formation of different levels of intracellular ice crystals: A. the formation of primary crystals with their diffusion layers within the intact intracellular solution; B. the formation of secondary or higher level crystals between the primary crystals.

Fig. 5

A schematic of the formation of different levels of intracellular ice crystals: A. the formation of primary crystals with their diffusion layers within the intact intracellular solution; B. the formation of secondary or higher level crystals between the primary crystals.

Fig. 6

Examples of intracellular vitrification in the presence of the extracellular crystallization when initial EG concentration is 5m and the NaCl concentration is: A. 0.3m; B. 0.45m; C. 0.6m.