During cryopreservation of biological material, the stage of formation of a solid phase (ice) during cooling of living cells and a cryoprotective mixture is of great importance. The formation of ice, along with other factors, significantly affects the survival of cells during the freezing–thawing process. Most researchers limit their studies to a temperature range of 0 to –80°C. This range is considered the most critical due to damage to biological material caused by intracellular and extracellular ice crystal formation. However, the processes that occur during further cooling of the frozen material to storage temperatures (–196°C) are poorly understood. When cooled below –100°C, thermomechanical stress in the frozen medium increases due to its compression, thus causing cracking. Exposure to ultrasound at the initial stage of freezing eliminated supercooling in all studied solutions and significantly altered the temperature curve. This study demonstrates the effect of ultrasound in the 0.88–8.00 MHz frequency range on the cracking of frozen cryoprotective solutions and the formation of ice microparticles. Freezing was performed in a thin 0.2-mm layer at cooling to –196°C. The shape and size of the ice microparticles were determined by cryomicroscopy. With increasing frequency, the degree of homogeneity (size and area) of the microparticles and the shape factor (degree of roundness) increased. It has been shown that ultrasound can effectively influence the formation of cracks in frozen cryoprotective media, especially in the high-frequency range. The greatest effect of increasing the degree of roundness of microparticles was observed in a medium containing saline solution, 10% dimethyl sulfoxide, and 10% egg yolk.
Andreev et al. (Mon,) studied this question.