Mechanistic Study Of Intracellular Ice Formation And Application

Direct cell injury in cryobiology is highly dependent on intracellular ice for-mation (IIF) during tissue freezing and thawing. Study of the intracellular iceformation (IIF) and growth (IIG) is essential to the mechanistic understandingof cellular damage by freezing. With the aid of high speed and high resolutioncryo-imaging technology, the transient IIF intracellular ice formation and growthprocesses of the attached human umbilical vein endothelial cells (HUVEC) andtumor cells (MCF-7) were successfully captured during freezing. It was foundthat the intracellular ice nucleation site was on the cell membrane closer to thenucleus. The ice growth was directional and toward the nucleus, which coveredthe whole nucleus before growing into the cytoplasm. The crystal growth ratein the nucleus was much larger than that in the cytoplasm, and its morphologywas in?uenced by the cooling rate. During the thawing process, small crystalsfused into larger ones inside the nucleus. Moreover, the cumulative fraction ofthe HUVEC with IIF was mainly dependent on the cooling rate but not the con-?uence of the cells attached. For MCF-7 cells, when frozen without ice seeding,IIF was observed to occur over a very small range of temperature ( 3 C). Thecrystal dendrites were indistinguishable, which was independent of cooling rate.Recrystallization was observed at the temperature from -13 C to -9 C duringthawing. On the contrary, IIF occurred from -7 C to -30 C when ice was seededat a high subzero temperature (i.e. -2.5 C). The morphology of intracellular icefrozen with ice seeding was greatly a?ected by the cooling rate, and no‘darken-ing’type ice formed inside cells during thawing. In addition, the intracellular iceformation was directional, starting from the plasma membrane and grew towardthe cellular nucleus with or without ice seeding. Ice crystal grew much faster and covered the whole intracellular space in comparison to that with ice seeding,during which ice stopped growing near the cellular nucleus. The results could beused to interpret findings in in-vivo cryosurgery of tumor, especially the cause ofinsuffcient killing of tumor cells in the peripheral area near vessels.In order to estimate the possibility of IIF, a set of models predicting thediffusion-limited ice nucleation and growth inside biological cells were built inthis study. Both heterogeneous and homogeneous nucleation mechanisms wereconsidered in the models. Molecular mobility including viscosity and mutualdiffusion coeffcient of aqueous cryoprotectant (i.e., glycerol here) solutions wasestimated using models derived from the free volume theory for glass transition.After being veriffed with experimental data, the models were used to predictthe critical cooling rate as a function of the initial glycerol concentration in anumber of cell types with different sizes. For slow freezing, it was found thatthe required critical cooling rate was cell type dependent with inffuences fromcell size, the ice nucleation and water transport parameters. For vitriffcation,it was further found that the thermodynamic and kinetic parameters for intra-cellular ice formation associated with different cells rather than the cell size perse signiffcantly affect the critical cooling rates. The model was then applied tothe study on the events of water transport and IIF during freezing mammalian(Peromyscus) oocytes. Unusual high activation energy for water transport wasidentiffed indicating water transport across the cell membrane was sensitive tocooling rate. The kinetics of IIF without ice-seeding was found to be stronglytemperature dependent. These ffndings are important for developing optimalvitriffcation protocols for cell cryopreservation.