Zhu Wenxin, Pan Ping'an, Huang Yonghua, Chen Wei, Han Sha, Li Zheng, Cheng Jinsheng
Institute of Refrigeration and Cryogenics, Shanghai Jiao Tong University, Shanghai 200240, P. R. China.
Department of Andrology, Center for Men's Health, Institute of Urology, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai 200080, P. R. China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2023 Oct 25;40(5):973-981. doi: 10.7507/1001-5515.202305004.
Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.
超快速冷却和复温速率是在冷冻和融化过程中实现无冰晶细胞的关键技术方法。基于蓝宝石薄膜开发了一套超快速固体表面冻融可视化系统，并在不同的冷冻保护剂成分、体积和激光能量下进行了液滴冻融实验。结果表明，1 μL混合冷冻保护剂[1.5 mol/L丙二醇(PG)+1.5 mol/L乙二醇(EG)+0.5 mol/L海藻糖(TRE)]的冷却速率可达9.2×10 °C/min。1-8 μL液滴的体积范围可以实现玻璃化。在比较多种冷冻保护剂的比例后，等比例混合渗透保护剂与海藻糖的组合具有最佳的玻璃化冷冻效果和更均匀的结晶特性。在复温操作过程中，首次测量了含金纳米颗粒的玻璃态液滴在400-1 200 W激光功率作用下的加热曲线，复温速率高达10 °C/min量级。根据不同功率复温过程的液滴图像，明确了具有微米级分辨率的无冰复温激光功率范围为1 400-1 600 W。本文的工作同时实现了液滴的超高速升温、瞬态视觉观测和温度测量，为冻融过程中判断无冰液滴提供了技术手段。有利于推动生物细胞和组织超快速冻融技术的发展。
