Modeling and typical cases analyze at the cell-scale of transmembrane transport and intracellular crystallization and recrystallization during the freeze-thaw process.

Abstract

Mechanical and solute damage caused by ice crystals during the freeze-thaw process of biological samples in cryopreservation are principal determinants of their activity. In this study, a numerical model is constructed by comprehensively considering the phenomenon of crystallization during cooling, recrystallization during rewarming, and the transmembrane transport of water and cryoprotective agent (CPA). The computational findings of the model demonstrate that higher cooling rates result in an increased volume of intracellular crystallization, with a correspondingly elevated intracellular nucleation temperature. By integrating the trend of CPA concentration variation during the cooling process, it is determined that the rates of 0.5 °C·min^-1 and 1 °C·min^-1 inflict minimal harm to mouse oocytes. During the rewarming process, the rate influences the intracellular ice volume, specifically the higher the rate of rewarming the smaller the increase in intracellular ice volume, and it is recommended that a high-power pulse be added before recrystallization to reduce the effects of recrystallization in practical applications. The pick-and-place operation of the cryopreservation vials can lead to recrystallization, and based on the calculations, it is recommended that the cryopreservation temperature should be lower than -160 °C and the operation time should be controlled within 90 s. The parameter scanning showed that a cooling rate of 0.4∼1.8 °C·min^-1 and an initial DMSO concentration of 0.1∼0.3 M are more favorable for the efficient recovery in the water bath of mouse oocytes. The model constructed in this study can provide valuable numerical guidance for practical cryopreservation protocols of biological specimens.