微液滴自然蒸发加载低温保护剂的细胞渗透特性与损伤评估  

Evaluation of Cell Permeability Characteristics and Damage during Natural Evaporation of Cryoprotectant-loaded Microdroplets

作　　者：刘林峰 崔梦冬 占太杰 何伟 胥义[1] Liu Linfeng;Cui Mengdong;Zhan Taijie;He Wei;Xu Yi(Institute of Biothermal Science and Technology,University of Shanghai for Science and Technology,Shanghai Tech-nical Service Platform for Cryopreservation of Biological Resources,Shanghai Collaborative Innovation Center for Tumor Treatment with Energy,Shanghai,200093,China)

机构地区：[1]上海理工大学生物系统热科学研究所、上海市生物资源低温保存技术服务平台上海市肿瘤能量治疗技术与器械协同创新中心,上海200093

出　　处：《制冷学报》2023年第6期156-166,共11页Journal of Refrigeration

基　　金：国家自然科学基金(52076140)资助项目。

摘　　要：本文提出了一种微液滴自然蒸发加载低温保护剂的方法,借助图像处理技术,结合细胞渗透模型与损伤模型进行理论分析,系统研究了初始体积和初始浓度对微液滴自然蒸发加载低温保护剂过程的细胞渗透特性与损伤的影响,并与传统低温保护剂加载方法(一步加载、两步加载)进行对比。研究结果表明:微液滴的初始体积越小,蒸发速率越快,低温保护剂更易达到目标浓度,能够有效缩短加载时间(1μL相比5μL缩短4907 s);随着低温保护剂初始浓度的降低,加载时间逐渐增长(0.5 M和2.5 M分别为2460、1346 s),但造成的细胞体积变化极值显著减小(0.5 M和2.5 M分别为初始体积的14%和43%),累积毒性损伤也会显著降低(0.5 M和2.5 M分别为23.05、47.53);微液滴自然蒸发加载相比传统加载方法可显著提高细胞存活率与细胞增殖能力(P<0.05),其细胞存活率为96.73%±0.54%,比一步加载提升16%,且培养48 h的细胞贴壁情况显著优于两步加载。表明该新方法能够简化低温保护剂加载流程,并能有效降低细胞损伤。Natural evaporation of microdroplets was studied as a new method for the addition of cryoprotectants.The effects of the initial volume and concentration on cell permeation characteristics and damage during cryoprotectant loading by natural evaporation of microdroplets were investigated systematically using image processing techniques.Combined with theoretical analysis of cell permeation and damage models,the results were compared with traditional cryoprotectant loading methods(one-step and two-step loading).The results show that,the faster the evaporation rate,the easier it was for the cryoprotectant to achieve the required concentration,which could reduce the loading time(by 4907 s for 1μL compared with 5μL).As the initial concentration of the protectant decreased,the loading time increased gradually(2460 and 1346 s for 0.5 M and 2.5 M,respectively);however,the cell volume change caused by extremes was significantly reduced(14%and 43%of the initial volume for 0.5 M and 2.5 M,respectively),as was the cumulative toxic damage(23.05 and 47.53 for 0.5 M and 2.5 M,respectively).Compared with the usual loading approach,microdroplet loading by natural evaporation considerably enhanced cell survival and proliferation(P<0.05).Cell survival was 96.73%±0.54%,a 16%increase over one-step loading,and cell wall adherence was considerably better at 48 h than that with two-step loading.This innovative technique substantially reduced cell damage while simplifying the cryoprotectant loading process.

关 键 词：低温保存 低温保护剂 液滴蒸发 渗透损伤 毒性损伤 

分 类 号：R318.52[医药卫生—生物医学工程] Q27[医药卫生—基础医学]