In Vitro Generation Of CIK Cells From The Cord Blood Mononuclear Cells And The Experimental Research Of Their Anti-tumor Effect

Objective: To build the experimental basement for the clinical use of CIK cells in tumor adoptive cellular immunotherapy, we established an effective protocol for their culture in vitro and explained their bio-characteristics. Methods: In order to obtain the CBMNCs , fresh aseptic cord blood was isolated by ficoll density gradient centrifugation. After washing, the CBMNCs were resuspended in complete RPMI1640 and induced into CIK cells and LAK cells at the presence of various cytokines such as IFN-r, IL-2, IL-1 and OKT3 at special times respectively. At the same time set the CBMNCs as the blank control group, which were not added any cytokines during the whole culture. An aliquot of cells were removed from the culture at various time points. The phenotypes, cellular cycles and the apoptoses of target cells were identified by flow cytometric analysis. At the proliferation peak, the cytotoxicity of CIK cells and LAK cells was determined by classic MTT assay. Results: According to the experiment, combining use of four types of cytokines could generate a great deal of CIK cells possessing highly cytotoxicity. From day 4 CIK cells became to proliferate and reached the peak at day 12. During the whole period, the relative percentage of CD3+CD56+cells increased significantly from 0.14% to 22%, expanded 163 folds. The absolute number also expanded about 1300 times. Other phenotypes of CIK cells, such as CD3+, CD4+, CD8+ cells also expanded at different extent. Compared with LAK cells, which reached the proliferation peak at day 8 and then showed no evident proliferation. LAK cells expanded only 64 folds in relative percentage. The control cells (CBMNCs) showed no evident change of phenotypes and proliferation of any cells. CIK cells showed a higher antitumor activity on the tumor lines of K562, Hela and YTMLC than LAK cells and CBMNCs in vitro, and their cytotoxicity increased along with the increasion of the effect-target ratio which indicated by the MTT assay. There were significant differences among three groups. At the same time, Flow cytometric analysis showed CIK cells could induce targetcells to apoptosis. Conclusion: 1.Cord blood can serve as an important material for the generation of CIK cells 2. Combining use of four types of cytokines can generate a great deal of CIK cells, and their proliferation peak appear around day 12, which is a suitable time for their clinical use 3. Inducing target cells to apoptosis is one of CIK cells' working ways 4. Among our study range of the concentrations: CIK cells possessing higher cytotoxicity in vitro than classic LAK cells, and so using CIK cells reasonably will provide a new hope for the adoptive cellular immunotherapy of tumor.