基于固体表面冷冻激光复温法的液滴冻融实验研究  

Dropletfreeze-thawingg system based on solid surface vitrification and laser rewarming

作　　者：朱文欣 潘平安 黄永华[1] 陈威[2] 韩厦 李铮[2] 程锦生 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;Shanghai Tofflon Medical Equipment Co.,Ltd,Shanghai 201108,P.R.China)

机构地区：[1]上海交通大学制冷与低温工程研究所,上海200240 [2]上海交通大学附属第一人民医院,泌尿外科中心男科男性健康评估中心,上海200080 [3]上海东富龙医疗装备有限公司,上海201108

出　　处：《生物医学工程学杂志》2023年第5期973-981,共9页Journal of Biomedical Engineering

基　　金：国家重点研发计划(2022YFC2703000);上海市科委创新行动计划(20S31903400);上海交通大学医工交叉研究基金(YG2021QN86)。

摘　　要：高升降温速率是实现细胞无冰冻融的关键技术途径。本研究研制了一套基于蓝宝石的超快速固体表面冷冻-激光复温冻融可视化系统,开展了不同冷冻保护剂组分和体积以及不同激光能量下的液滴冻融实验研究。结果表明:冷冻操作时该系统可实现1μL体积混合低温保护剂[1.5 mol/L丙二醇(PG)+1.5 mol/L乙二醇(EG)+0.5 mol/L海藻糖(TRE)]的降温速率高达9.2×10^(3)℃/min,且1~8μL容积范围的液滴均可实现玻璃化冷冻;经过比对多种冷冻保护剂配比浓度,等比例混合渗透性保护剂和海藻糖的组合具有最好的玻璃化冷冻效果和更均匀的结晶特性。在复温操作时,首次测得400~1 200 W激光功率作用下含有金纳米颗粒的玻璃态液滴的升温曲线,速率高达106℃/min量级。依据不同功率复温过程的液滴图像,明确了实现微米级分辨率无冰复温的激光功率范围为1 400~1 600 W。本文工作同步实现了液滴的超高速升降温、瞬态可视化观察和温度测量,为判断液滴无冰冻融提供了技术手段,有利于促进生物细胞和组织超快速冻融技术的发展。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 1μL 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^(3) ℃/min.The volume range of 1-8μL 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 400-1200 W laser power,and the rewarming rate was up to the order of 10^(6)℃/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 1400-1600 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.

关 键 词：细胞低温保存 玻璃化 固体表面冷冻 激光复温 

分 类 号：TB34[一般工业技术—材料科学与工程] R318.52[医药卫生—生物医学工程]