Influence of droplet size to cell viability in flash freezing method

Abstract

We examine cell cryopreservation method without cryoprotectant agent (CPA). In general, the CPA is supplemented to the freezing medium to inhibit the generation and growth of ice crystals inside and outside the cell because cell structures are destroyed by the ice crystals. On the other hand, we need to think about the cytotoxicity of CPA in the cell cryopreservation. The faster the cooling rate become, the more the generation and growth of ice crystals are inhibited inside and outside the cell. To freeze picolitter droplets at high cooling rate, we used inkjet technique and liquid nitrogen. Thereby, the damage to the cells was inhibited. The viability of the flash frozen cells at 20 pL improved about 60 times higher than the viability of frozen cells by slow-rate freezing without CPA. We confirmed that the cell viability of cryopreservation method without CPA increased in flash freezing. Moreover, the viability of the flashly frozen cells at 20 pL increased to more than twice the viability of the flash frozen cells at 200 pL. As the droplet size became small, the cell viability increased in the flash freezing method. We were able to find the possibility of the inkjet-based flash cell freezing toward cryopreservation without CPA.