Micro-droplet spray vitrification system produces small droplets and can be vitrified at a lower concentration of cryoprotectant (CPA). In this study

HepG2 cells were vitrified using micro-droplet vitrification system; moreover

damages caused by CPA loading

spraying

receiving

and vitrification/rewarming processes were evaluated. In addition

CPA formulations were optimized by reducing the Me2SO concentration and adding an appropriate amount of trehalose. The results showed that each step in micro-droplet spray vitrification may cause damage to the cells. CPA loading

spraying

vitrification and rewarming processes caused severe damages

but mild damages were caused by the sheet receiving process. Cell activities after cryopreservation are significantly reduced with a decrease in the Me2SO concentration. When the CPA formulations are constant

cell activities in the vitrification group become better than those in the slow freezing group. Moderate amounts of trehalose can enhance the effect of cryopreservation

while excessive trehalose can be counteractive. When cells were preserved with 5% Me2SO + 0.3M trehalose as a cryoprotectant

the cell survival rate reached (92.42±0.95)% and the 24 h adherence rate reached (95.64±1.03)%; therefore

the droplet spray vitrification effect was found to be the best.