Method Optimization For Isolation Of Mononuclear Cells And Induction And Culture Of CIK Cells

Cytokine induced killer(CIK),which is induced by cytokines,is a cytotoxic T cell,which is induced by a variety of cytokines such as interleukin-2(IL-2),interferon(IFN)and CD3 monoclonal antibody.In recent years,with the development of modern medicine,medical technology has made great progress.The new therapy based on biotechnology,especially cell therapy,has developed rapidly,and the application of CIKcell therapy is more extensive.Equal density centrifugation is a technical scheme widely used to enrich specific cells in cell biology.Because the density of the selected liquid medium is equal to or slightly larger than the intended cell,the target cell will sink the interface of the liquid medium into centrifugation regardless of the length of the centrifuge,regardless of the length of the centrifuge.The bottom of the container is further enriched at the interface of the liquid medium(i.e.the equilibrium position).A typical equal density centrifugation method uses a single density gradient centrifugation to purify the lymphocytes in the blood or bone marrow samples,and the liquid medium is usually a aqueous solution with a physiological osmotic pressure containing a combination of polysucrose and iodide.In this study,we collected umbilical cord blood samples to study the yield and purity of mononuclear cells and CIKcell yield,optimize the isolation of mononuclear cells,and systematically optimize the preparation of CIKcells.We have finished the following work.1.Establish a library of umbilical cord blood samples.According to the different umbilical cord blood volume,nearly 30 neonatal umbilical cord blood samples were selected,including 10 samples with umbilical cord blood volume less than 50 m L,10samples with blood volume between 50 m L and 100 m L,and 10 samples with blood volume greater than 100 m L.The above samples were grouped to investigate the effect of different sample sizes on the isolation of mononuclear cells,which laid a solid foundation for the subsequent optimization of isolation conditions and CIKcell induction conditions.2.Density centrifugation was used to collect mononuclear cells from cord blood.It was found that Ficoll-Paque^TM PLUS was superior to Nyco Prep^TM 1.077 in isolation of mononuclear cells,and had no significant difference with the self-made isolation medium.The greater the yield and purity of mononuclear cells,the higher.3.CIKcells were induced and cultured,3*10^6 cells were added into 6-well plate,serum-free medium containing 1 000 U/m L INF-gamma was added,cultured at 37 C for 24 hours with 5%CO2,added 50 ng/m L anti-CD3 and 300 U/m L IL-2,and continued to be cultured.After 3 days,the cells were sub-cultured with 3*10^6 cells per hole and supplemented with IL-2 to 300 U/m L.Flow cytometry analysis showed that the ratio of umbilical cord blood volume to blood preservation solution had no significant effect on the purity of CIKcells.According to these findings,the combination of hydroxyethyl starch and sodium diatrizoate could be more easily used for clinical treatment.Increasing the ratio of umbilical cord blood volume to blood preservation solution could improve the yield and purity of mononuclear cells,and then induce more CIKcells to be cultured for treatment,which could be improved significantly.The therapeutic effect is of great significance to clinical application.