外周血液样本抗凝剂类型、保存时间和温度对CIK细胞培养的影响  

Selection of storage time,temperature and anticoagulants of peripheral blood samples for culturing cytokine-induced killer cells

作　　者：王宇环 马飞 付凡 魏宗科 董志瑜 朱利军 罗晓玲 

机构地区：[1]深圳市合一康生物科技股份有限公司,广东深圳518045

出　　处：《细胞与分子免疫学杂志》2016年第3期382-386,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：深圳市科技计划项目(CXZZ20140509144527788)

摘　　要：目的探讨外周血液样本抗凝剂类型、保存时间和保存温度对细胞因子诱导的杀伤（CIK）细胞培养的影响,为自体免疫细胞治疗技术的外周血保存标准化提供实验依据。方法将抽取的4份60 m L外周血样本分别应用肝素钠、细胞保存液（枸橼酸钠）和EDTA抗凝剂抗凝,并分别存放于4、22、30℃的环境中,储存0、4、8、24 h,应用正交实验设计优选为12组,然后分离出单个核细胞分别诱导培养成CIK细胞,对比12组CIK细胞增殖效率及γ干扰素（IFN-γ）分泌量的变化。结果在CIK细胞培养过程中,EDTA组CIK细胞增殖不明显,肝素钠组和血液保存液组CIK细胞增殖明显,且肝素钠优于血液保存液;血液保存时间对CIK细胞培养影响不明显,但保存时间越长,细胞增殖效率越差,保存时间超过8 h的CIK细胞,其增殖效率在第16天以后明显降低。对培养时间在16 d内的CIK细胞,血液保存温度影响不显著,但CIK细胞培养16 d之后,22℃保存条件下,CIK细胞增殖效率最高,4℃次之,30℃最差。结论肝素钠和血液保存液均可用于CIK细胞培养的血液抗凝;保存时间在24 h内,温度在4℃~30℃之间保存的血液,均可用于CIK细胞培养。Objective To investigate the effects of anticoagulants,preservation time and temperature of peripheral blood samples on the culture of cytokine-induced killer（ CIK） cells so as to provide the experimental evidences for peripheral blood storage in autologous immunotherapy. Methods Four samples of 60 m L peripheral blood were collected. After being added with heparin sodium,cell preservation liquid（ sodium citrate） and EDTA solution,they were separately stored under 4℃,22℃,and 30℃ for 0,4,8,24 hours. We divided the orthogonal experiments into 12 groups,and then separated mononuclear cells and induced them into CIK cells. The proliferation efficiency and IFN-γ secretion were compared in the 12 groups.Results The proliferation of CIK cells was not obvious in EDTA group,but obvious in heparin sodium group and sodium citrate group,especially better in sodium citrate group. The storage time of blood did not have a significant impact on CIK cell culture,however the longer storage time, the lower cell proliferation efficiency. The proliferation efficiency decreased apparently after 16-day culture if preservation time exceeded 8 hours. After the culture period of 16 days,the efficiency of CIK cell proliferation was the highest at 22℃,followed by that at 4℃,and the lowest was at 30℃. It was not apparent that the temperature of blood storage affected CIK cell proliferation within 16-day culture period. Conclusion Both heparin sodium and sodium citrate can be used in blood sample anticoagulation for CIK cell culture. Blood samples are suitable for CIK cell culture which are stored within 24 hours between 4℃ and 30℃.

关 键 词：细胞因子诱导的杀伤细胞 抗凝剂 保存温度 保存时间 

分 类 号：R446.11[医药卫生—诊断学] R392.12[医药卫生—临床医学]