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Archive > Volume 20 Issue 3 > 2013,20(3):342-347. DOI:10.3872/j.issn.1007-385X.2013.03.015 Prev Next
Technigue and Method
Effect of cryopreservation on bioactivity of cytokine induced killer cells derived from human cord blood after in vitro culture of different time
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Abstract:
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 (\[35.90±1.67\]×106, \[18.98±2.13\]×106, and \[11.76±2.12\]×106, 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.
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