In slow-freezing, cells in a medium are cooled to below freezing point. At some stage, ice
masses containing pure crystalline water will form. What remains between the growing ice
masses is the so-called unfrozen fraction, in which all cells and all solutes are confined (see Figure 7). The concentrations of sugars, salts and cryoprotectant (e.g. glycerol) increase, while the
volume of the unfrozen fraction decreases. The increase in osmotic strength causes an efflux
of water from the cells. Slow cooling is needed in order to allow sufficient efflux of water to
minimize the chance of intracellular ice formation. As cooling continues, the viscosity of the
unfrozen fraction ultimately becomes too high for any further crystallization. The remaining
unfrozen fraction turns into an amorphous solid that contains no ice crystals.