一种新贴壁细胞冻存液的研发与评价  

Development and evaluation of a novel cryopreservation medium for adherent cells

作　　者：刘思景[1] 李雪[2] 张咏怡 李柳郡 邱峰 梅晓雯 余佳蓓 秦笙[1] LIU Si-jing;LI Xue;ZHANG Yong-yi;LI Liu-jun;QIU Feng;MEI Xiao-wen;YU Jia-bei;QIN Sheng(Department of Laboratory Medicine,Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,Guangdong,China;Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China;Department of Laboratory Medicine,Nanhai Hospital,Southern Medical University,Foshan 528244,Guangdong,China)

机构地区：[1]广东省中医院检验医学部,广东广州510120 [2]广州中医药大学,广东广州510006 [3]南方医科大学南海医院,广东佛山528244

出　　处：《生物医学工程与临床》2022年第6期677-681,共5页Biomedical Engineering and Clinical Medicine

基　　金：广东省自然科学基金资助项目(2021A1515011507);广东省中医院中医药科学技术研究专项课题(YN2019QL05);广东省佛山市卫计局医学科研课题(20190282);广州中医药大学第二临床医学院创新基金资助项目(20201813F1;20201824F2)。

摘　　要：目的评价一种新的贴壁细胞冻存液。方法使用含7.5%二甲基亚砜(DMSO)(V/V%)、17.5%丙二醇(V/V%)、2.5%聚乙二醇(V/V%)、10%胎牛血清(FBS)(V/V%)的达氏修正依氏培养液(DMEM)作为新型冻存液,对MDCK、LLC-MK2、HEp-2等3种贴壁细胞进行冻存实验。实验分常规冻存组、贴壁细胞冻存对照组和贴壁细胞冻存组,通过观察细胞形态并以HEp-2细胞为典型绘制细胞生长曲线;利用细胞病变法及免疫荧光法检测复苏的贴壁细胞对甲型流感病毒、副流感3型病毒、呼吸道合胞病毒的敏感性变化。结果3种贴壁细胞冻存组复温后细胞保持完整的单层贴壁状态,与常规冻存组细胞复苏培养3 d后的新鲜贴壁细胞形态一致;3种贴壁冻存对照组细胞复苏后其细胞大量死亡,数量急剧减少,细胞间隙增大,细胞呈破碎样。生长曲线结果显示HEp-2贴壁细胞冻存组一直保持5×10^(5)/mL水平。对上述病毒的敏感性检测中,贴壁细胞冻存组呈现出典型的细胞病变;感染甲型流感病毒的MDCK贴壁冻存细胞荧光呈胞浆颗粒型或细胞核均质型;感染呼吸道合胞病毒的HEp-2贴壁冻存细胞荧光呈胞浆颗粒型,合胞体中亦可见小块包涵物染荧光;感染副流感3型病毒的LLC-MK3贴壁冻存细胞荧光呈胞浆颗粒型强绿色荧光。结论新研发的贴壁细胞冻存液可用于多种贴壁细胞的原位冻存,能保持细胞活力和对病毒的敏感性,为大批量即用型细胞产品的开发应用奠定基础。Objective To evaluate a novel cryopreservation medium for adherent cells.Methods The Dulbecco’s modified Eagle’s medium(DMEM)was used as a novel freezing medium,which contained 7.5%dimethyl sulfoxide(DMSO)(V/V%),17.5%propylene glycol(V/V%),2.5%polyethylene glycol(V/V%)and 10%fetal bovine serum(FBS)(V/V%),and 3 kinds of adherent cells(MDCK,LLC-MK2 and HEp-2)were cryopreserved.Routine cryopreservation group,adherent cell cryopreservation control group and adherent cell cryopreservation group were set up for the experiment.The cell morphology was observed,and cell growth curve was drawn with HEp-2 cells as the typical example;the susceptibility of resuscitated adherent cells in influenza virus A,parainfluenza type 3 virus and respiratory syncytial virus were detected by cytopathic assay and immunofluorescence assay.Results The cells in the 3 types of adherent cell cryopreservation groups maintained the complete monolayer adherent state after rewarming,which was consistent with the morphology of fresh adherent cells in routine cryopreservation group after3-day of recovery.After cell resuscitation,a large number of cells in 3 types adherent cell cryopreservation control group became apoptotic and the number of cells decreased sharply,the intercellular space increased,and the cells were broken.The growth curve showed that the HEp-2 adherent cell cryopreserved group maintained level of 5×10^(5)/mL.In virus susceptibility test,the adherent cell cryopreserved group showed typical cytopathic changes;MDCK adherent cryopreserved cells infected with influenza virus showed cytoplasmic granular or nuclear homogeneous fluorescence;HEp-2 adherent cryopreserved cells infected with respiratory syncytial virus showed cytoplasmic granular fluorescence,and small inclusions were also found in the syncytium and stained with fluorescence;LLC-MK3 adherent cryopreserved cells infected with parainfluenza type 3 virus showed strong green fluorescence of cytoplasmic granular.Conclusion It is demonstrated that the novel developed adherent

关 键 词：贴壁细胞 冻存液 病毒感染 冻存实验 MDCK细胞 LLC-MK2细胞 HEP-2细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]