Research on the Cryopreservation System of Controlled Rapid Cooling and Rewarming of Cellular Micro-Droplets

The vitrification preservation methods for cell microdroplets have received considerable attention in the field of cryopreservation. However, the vitrification process for microdroplets has limitations such as uncontrollable cooling/rewarming processes. In this study, a cryopreservation system for controllable rapid cooling and rewarming during the vitrification of cell microdroplets was constructed by combining the lifting of a moving slide table with the Joule heating rewarming method. This system achieves rapid and controllable cooling by adjusting the speed of immersion in liquid nitrogen and realizes a fast rewarming process by controlling the heating time and current intensity of Joule heating. Thus, it realizes the vitrification of microdroplets and significantly prevents damage to cells caused by devitrification during the droplet rewarming process. The results show that the cooling rate controlled by this system attained 1.8 × 10⁴ ℃/min. This enabled the vitrification preservation of cell microdroplets with a relatively low concentration of cryoprotectant. The rewarming rate control attained 4.0 × 10⁴ ℃/min. This effectively prevented the occurrence of devitrification and ice crystal regrowth during the rewarming process of the relatively low concentration of cryoprotectant. Cryopreservation of A549 cells in microdroplets verified that the survival rate after cryopreservation and resuscitation using this system is significantly higher than that after conventional straw preservation. The research presented in this paper is likely to provide new solutions for the automatic vitrification preservation and rewarming of cell microdroplets.