Experimental Research On Biological Characteristics And Anti-tumor Activity Of CIK Cells Stimulated By RetroNectin

ObjectiveComparison of traditional culture CIK cells with a new method including RetroNectin in proliferation activity,increasing proportion of the main effective cells and cytotoxicity of cytokine induced killer(CIK) cells.To establish a highly effective simple and convenient method of culture CIK cell.MethodsPeripheral blood mononuclear cells(PBMCs) were separated from a component of white blood cell(WBC) of 10 healthy person through density gradient centrifugation with the lymphocyte-separating medium.Half isolated PBMCs were cultured in serum-free medium KBM551,supplemented with RetroNectin and CD3 McAb and IL-2.Cultured in an incubator with 5%CO2 in air at 37℃.Supplemented with IL-2 only for future culture.A half dose of the medium was changed every 3 days.The other half isolated PBMCs were cultured in serum-free medium KBM551 supplemented with CD3 McAb,IL-2,IFN-γand IL-1α.Then the changes in the proliferation activity and phenotypes of the cells were observed.The cells were harvested after 15 days.MTT assays were used to determine the cytotoxicity in vitro.TH1 cytokines(IFN-γ) and TH2 cytokines(IL-4),perform and granzymeB were identified by flow cytometric analysis.ResultsCompared with traditional culture CIK cells the new method including RetroNectin has more effeciency in proliferation activity,increasing proportion of the main effective cells and cytotoxicity of cytokine induced killer(CIK) cells.After cell culture,the percentage of CD3+,CD3+CD8+,CD3+CD16+56+cells increased greatly. (CD3+,96.99±4.63%),(CD3+CD8+,71.34±8.53%),(CD3+CD16+56+,28.83±6.54%), and the percentage of CD3+CD4+cells decreased obviously,(CD3+CD4+, 16.81±7.44%).The ratio of effective CIK cells to target cells were 20:1 and 40:1.The mean percent specific lysis of A,B group cells towards Hela,K562,A549,G823 was: A group:32.47±7.51%and 55.15±8.43%(Hela),48.43±8.15%and 65.75±8.39%(K562), 58.58±8.52%and 65.73±14.79%(A549),67.54±18.37%and 74.28±15.71%(G823).B group:45.30±5.16%and 65.43±4.91%(Hela),47.16±6.13%and 62.90±7.25%(K562), 70.8±11.23%and 83.44±13.76%(A549),75.10±7.24%and 88.6±12.28%(G823).The level of the secreted TH1 cytokines IFN-γ,perforin,granzymeB were higher than in PBMCs.ConclusionAfter cell culture,the percentage of CD3 +,CD3 + CD8 +,CD3 + CD16 +56 + cells increased greatly,and the percentage of CD3 + CD4 + cells decreased obviously. The level of the secreted TH1 cytokines IFN-γ,perforin,granzymeB in cultured cells were significantly higher than in PBMCs.CIK cells from both didn't have obvious differences in henotype,cytotoxicit.But for the advantage of fewer Peripheral blood, lower opportunity infection and fewer activate facts,the new method of culture CIK cells should be adopted.