微滴喷射玻璃化的过程损伤分析及保护剂优化

Process Damage Analysis and Cryoprotectant Optimization for Micro-droplet Spray Vitrification

微滴喷射玻璃化保存系统产生的微滴尺寸较小,在较低浓度的低温保护剂条件下即可实现玻璃化。本文采用微滴喷射玻璃化保存系统对HepG2细胞进行玻璃化保存,研究保护剂加载过程、喷射过程、接收过程及玻璃化/复温过程对细胞造成的损伤程度,并通过降低保护剂中Me2SO浓度、添加适量海藻糖来优化保护剂配方。结果表明,微滴喷射玻璃化保存各过程对细胞均有损伤,保护剂加载过程、喷射过程、玻璃化及复温过程对细胞造成的损伤较大,薄片接收过程对细胞造成的损伤小。另外,随着保护剂中Me2SO浓度的降低,低温保存后的细胞活性明显降低;保护剂浓度相同时,玻璃化保存效果较慢速冷冻效果好;适量的海藻糖能够起到增强低温保存效果的作用,过量则起到降低作用;以5%Me2SO+0.3 mol/L海藻糖作为低温保护剂玻璃化保存细胞时,细胞存活率达(92.42±0.95)%,24 h贴壁率达到(95.64±1.03)%,微滴喷射玻璃化效果最好。

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+0.3 mol/L 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.