Vitrification of Biological Cells Using a Cryogenic Fine Solid Particulate Spray

We have developed a cryopreservation method that uses a single-component super-adiabatic two-fluid de Laval nozzle for the continuous production of solid nitrogen particles with a micro-nano-order particle size. The cell viability results showed that use of the solid nitrogen spray improved cell viability at the time of thawing by more than 20% compared with the conventional liquid nitrogen immersion freezing method. By using the present dynamic spray cooling method that avails of the synergistic effect of impingement heat transfer, convective heat transfer, and evaporation latent heat transfer of solid nitrogen particles, the new vitrification method reduces the cell membrane disruption caused by ice nucleation, crystal growth, and volume expansion.