ABSTRACT: In cytokine-induced killer (CIK) cell therapy, the phenotypes and the numbers of CIK cells have a great influence on the therapeutic effects. This study aimed to investigate the effects of different ex vivo cell culture methods on the proliferation and cytotoxicity of CIK cells that were obtained from gastric cancer patients.CIK precursor (Pre-CIK) cells were collected by either hydroxyethyl starch (HES) sedimentation (HES method, unpurified group) or Ficoll-Hypaque density gradient centrifugation (Ficoll method, purified group). Cell number, collection time, and morphology of Pre-CIK cells in the two groups were determined. The proliferation ability, cytokines, phenotypes, and cytotoxicity of CIK cells in the two groups were evaluated ex vivo and in vivo.In this study, the number of Pre-CIK cells in the unpurified group was significantly higher than that in the purified group (P<0.05). Numbers of erythrocytes, platelets, and granulocytes were reduced significantly following the purification step (P<0.05). Compared to CIK cells in the purified group, those in the unpurified group showed more active proliferation, accompanied by higher percentages of CD8+, CD3-CD56+, and CD3+CD56+ cells, which were responsible for cytotoxicity of CIK cells (P<0.05). This research also showed that the levels of interferon-?, interleukin-2, and tumor necrosis factor-?, which can enhance the proliferation and cytotoxicity of CIK cells, were significantly increased in the unpurified group (P<0.05). Furthermore, CIK cells in the unpurified group also showed stronger anti-tumor effects against gastric cancer cells than those in the purified group, both ex vivo and in vivo (P<0.05).The removal of Ficoll-Hypaque purification step reduces the time and cost of the Pre-CIK separation and provides more CIK cells with higher cytotoxicity, which is of great importance in the clinical application of CIK cell therapy.