Optimization Of CIK Cell Culture System And Its Killing Effect On Liver Cancer Cell Lines

Objectives:By comparing the amplification multiples of cytokine induced killer cells from umbi-lical cord blood and peripheral blood of tumor patients with different concentrations of IL-2 in the culture medium,a more suitable culture environment for the growth of CIK cells was found.And then,the proliferation indexes of the two types of CIK cells were compared under the same culture conditions,in order to find a stable and efficient culture method and screen a seed cell source with a large proliferation potential.By co-culturing two kinds of CIK and hepatocellular carcinoma cells,the in vitro efficacy of the two kinds of CIK agai-nst hepatocellular carcinoma cells was compared,and the the optimal action time and targ-et ratio of the two kinds of CIK against hepatocellular carcinoma cells were found.It prov-ides experimental basis for hepatocellular carcinoma cells immunotherapy and future clini-cal research.Methods:RPMI 1640 culture medium and GTT-551 culture medium,and the final concentr-ation of IL-2 was 0 IU/ml,200 IU/ml,and 500 IU/ml respectively in the culture system to induce culture of single nuclear cells derived from umbilical cord blood source and periph-eral blood source of tumor patients.Observed the morphology change of CIK cell under optical microscope,trypan blue was used to take counts of the number of cells and the growth curves was plotted,calculate the cell expansion factor.CCK8 assay was used to determine the trend of cell proliferation,and flow cytometry was used to determine the ph-enotypes of CD3 and CD56 of the two CIK cells after induction.On the 10th day of cultu-re,CIK cells from the two sources were co-cultured with hepatocellular carcinoma cell line-7721 to detect their killing effect in vitro.SPSS20.0 software was used for statistical analysis of the experimental data.Results:1.When using RPMI 1640 culture medium,the amplification multiples of umbilical cord blood CIK and patient peripheral blood CIK cells at different IL-2 concentrations were(19.7±3.5)vs(5.3±2.2)、(55.1±2.6)vs(31.5±4.1)、(92.9±8.9)vs(58.8±4.8),respec-tively(P<0.05).When using GTT-551 medium,the amplification multiples of umbilical cord blood CIK cells and patient peripheral blood CIK cells at different il-2 concentrations were(33.9±1.4)vs(13.0±4.2)、(87.7±3.8)vs(45.3±1.8)、(131.6±9.2)vs(84.7±7.8),resp-ectively(P<0.05).2.The proportion of CD3~+CD56~+cells was(27.2±4.7)%and(22.1±5.3)%(P<0.05),there was no significant difference in the ratio of CD3+CD56+cells between umbilical co-rd blood CIK and patient peripheral blood CIK cells after induction culture.3.After 24h co-culture of two CIK cells and hepatocellular carcinoma cells,the kill-ing rate of CIK in umbilical cord blood from low to high target ratio was(7.6±1.5)%vs(5.3±0.4)%、(14.5±2.0)%vs(6.0±0.3)%、(30.1±3.8)%vs(17.1±4.6)%、(46.4±3.3)%vs(31.5±5.7)%,respectively(P<0.05).After a total of 48h of culture,the killing rate of the two groups was(9.1±0.4)%vs(7.3±0.7)%、(21.9±2.2)%vs(12.4±1.1)%、(42.4±4.1)%vs(29.9±4.2)%、(58.8±4.8)%vs(44.5±3.2)%,respectively(P<0.05).After a total of 72h of culture,the killing rate of the two groups was(11.3±2.6)%vs(9.8±2.7)%、(33.5±3.8)%vs(25.1±4.6)%、(49.8±1.8)%vs(40.6±3.0)%、(66.7±2.8)%vs(55.6±2.5)%,respectively(P<0.05).Except for the peripheral blood CIK group,which was co-cultured for 24h,the eff-ect target ratio at 10:1 and 20:1 was P>0.05,and the rest were statistically significant.Conclusions:1.Both of the two culture medium can effectively promote the proliferation of CIK cells.The GTT-551 medium has a more significant effect on the proliferation of CIK cells from the two sources,it indicates that the GTT-551 culture medium is more suitable for CIK cell culture in vitro.The proliferation of CIK cells is concentration dependent on IL-2,the higher the concentration of IL-2,the higher the cell expansion factor.2.Umbilical cord blood has a faster expansion rate than peripheral blood of patients,but there is no significant difference in the proportion of CD3~+CD56~+cell phenotypes bet-ween them.3.The tumor killing effect of umbilical cord blood CIK was stronger,and the killing effect of CIK cells on hepatocellular carcinoma 7721 cells was positively correlated with the target ratio,the killing rate was highest when the effective target ratio was 80:1,and the optimum co-incubation time was 72h.