Cells and cell cultures
The generation of CIK cells was based on a previous study [22]. Blood (80 mL) was obtained from heparinized peripheral blood of all the patients. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation. Cells were cultured in X-15 medium containing 2% autologous serum and adhered for 1 hour. The suspension cells were stimulated with 1000 U/ml IFN-gamma for the first 24 hours, then induced into CIK cells with 100 ng/ml OKT-3, 1000 U/ml RHIL-2 and 100 U/ml IL-1α. At 14 days, the fraction of CIK cells were collected to assess their number, phenotype, and viability of cells, and to test for possible contamination by bacteria, fungi, or endotoxins. Vitalities of CIKs determined by trypan blue staining, CIKs activity and CD3+CD45+ T cells were accounted for more than 95% and 90% of total cells. Then, autologous CIK (1.0-1.5*1010 cells) were transferred to patients via intravenous infusion.