目的： 观察体外培养不同时间后冻存对人脐血来源细胞因子诱导杀伤（cytokine induced killer，CIK）细胞增殖、免疫表型、细胞毒活性以及细胞因子分泌的影响。 方法： CIK细胞在体外培养6 、9 、12 d后冻存1个月，复苏后培养至72 h，其间每隔24 h检测细胞增殖情况，并采用流式细胞术检测复苏后细胞免疫表型、CCK-8法检测复苏后细胞对A549细胞的杀伤活性、ELISA方法检测复苏后CIK细胞分泌细胞因子IFN-γ和TNF-α的变化。 结果： 体外培养不同时间后冻存的CIK细胞在复苏后仍然显示出较好的增殖活性，其中，体外培养6 d后冻存组CIK细胞增殖活性明显高于体外培养9 d和12 d后冻存组CIK细胞，复苏72 h时，6、9、12 d冻存组CIK细胞数分别为(35.90±1.67)×106、(18.98±2.13)×106和(11.76±2.12)×106个（P<0.01）。 体外培养6、9、12 d后冻存组CIK细胞复苏24 h后，各冻存组CD3+CD56+、CD3+CD8+细胞比例逐渐增加，且复苏72 h后6 d冻存组CIK细胞中CD3+CD56+和CD3+CD8+细胞比例最低。体外培养后冻存组CIK细胞在复苏24 h内对A549细胞的杀伤活性较低，但24 h后细胞毒活性有较大幅度的增加，且体外培养12 d后冻存组CIK细胞对A549细胞的杀伤活性高于6 d和9 d冻存组CIK细胞，如复苏后72 h，12、6、9 d冻存组CIK细胞对A549细胞的抑瘤率分别为（0.81±009）%、（0.59±0.06）%、（0.42±0.08）%（P<0.01）。ELISA检测结果显示，随着复苏时间的延长，体外培养不同时间后冻存的CIK细胞分泌IFN-γ和TNF-α的水平也逐渐上升；复苏72 h时，体外培养6 d后冻存组CIK细胞分泌IFN-γ的量要高于其他组，而体外培养12 d后冻存组TNF-α的分泌量则明显高于其他组。 结论： 体外培养不同时间后冻存对CIK细胞的生物学活性有一定影响，但复苏后经短时间培养后基本恢复，提示CIK细胞可以在培养至12 d后进行冻存。

Objective:To observe the effect of in vitro culture of different time on proliferation, immunophenotype, cytotoxicity and cytokine secretion of human cord blood derived cytokine induced killer (CIK) cells after cryopreservation.Methods: CIK cells were cryopreserved for 1 month after in vitro culture for 6, 9 and 12 d. The cell proliferation was detected every 24 h when they were recovered and cultured for 72 h. The immunophenotype, cytotoxicity on A549 cells and the secretion of IFN-γ and TNF-α of CIK cells after recovery were detected by flow cytometry, CCK-8 assay and ELISA assay, respectively. Results:CIK cells, in vitro culture of different time before cryopreservation still showed a superior proliferation activity after recovery, among which the proliferation of CIK cells in vitro cultured for 6 d before cryopreservation, was significantly higher than those in vitro cultured for 6 and 12 d before cryopreservation. The CIK cell counts were (\[35.90±1.67\]×106, \[18.98±2.13\]×106, and \[11.76±2.12\]×106, P<0.01) in 6, 9 and 12 d cryopreservation groups after recovery for 72 h. After recovery for 24 h, the proportions of CD3+CD56+ and CD3+CD8+ cells increased gradually in all the three 6, 9 and 12 d cryopreservation groups. Moreover, both two cell proportions in 6 d cryopreservation group after recovery for 72 h was the lowest. The cytotoxicity of CIK cells on A549 cells within 24 h of recovery in all the three cryopreservation groups was much low. However, their cytotoxicity increased considerably after 24 h. The cytotoxicity of CIK cells on A549 cells in 12 d cryopreservation group was higher than those in the 6 and 9 d cryopreservation groups. For example, the anti-tumor rate of CIK cells on A549 cells in 6, 9 and 12 d cryopreservation groups after recovery for 72 h was (\[0.81±0.09\]%, \[0.59±0.06\]% and \[0.42±0.08\]%, P<0.01). ELISA assay results showed that, the levels of IFN-γ and TNF-α secreted by CIK cells increased along the recovery time in all cryopreservation groups that had been in vitro cultured for various times. After recovery for 72 h, the level of secreted IFN-γ in 6 d cryopreservation group was higher than those of the other groups, and the level of secreted TNF-α in 12 d cryopreservation group was higher than those of the other groups.Conclusion:In vitro culture of different time can influence the bioactivity of cryopreserved CIK cells. However, the bioactivity can be restored after recovery for a short time, demonstrating that CIK cells can be cryopreserved after in vitro culture for 12 d.