AIM:To improve the preparation of adherent lymphokine-activated killer(A-LAK)cells and to study the effects ofcryopreservation and phenylacetate(PA)on biologicalcharacters of A-LAK cells.METHODS:A-LAK cells were obtained from peripheral bloodmo■onuclear cells(PBMCs)of the patients withhepatocellular carcinoma(HCC)by using L-phenylalaninemethyl ester(PME)to deplete immunosuppressivemonocytes.Proliferative activity of SMMC7721 cell line aftertreatment with phenylacetate(PA)was observed.A-LAKcells were treated with the supernatant of SMMC7721 cellsthat had been pretreated with PA.The changes ofproliferation,cytotoxicity and phenotype of A-LAK cellswere investigated after cryopreservation.RESULTS:The expansion of A-LAK cells(96.79±69.10 foldson Day 14)was significantly higher than that of non-adherent LAK(NA-LAK)cells(22.77±13.20)as well asconventional LAK cells(4.64±0.91).PA significantlysuppressed the growth of SMMC7721 cells,and the inhibitorratio was 46%.The supernatant of cultured tumor cellsintensively suppressed the proliferation and cytotoxicity ofA-LAK cells,but the suppressive effect of the supernatantwas previously decreased after treatment with PA.Impairments in proliferation and cytotoxicity of A-LAK cellsimmediately after thawing of cryopreservation and recoveryafter reincubation with IL-2 were observed.The cytotoxicityof thawed A-LAK cells on Day 5 was significantly higher thanthat of fresh A-LAK before freezing(54.8±10.2 % vs 40.5±6.4 %).No significant change in the percentage oflymphocyte subsets was identified in frozen A-LAK cells ascompared with that in the fresh control cells.CONCLUSION: A-LAK cells can be simply prepared by using PME. and showed a synergistic anti-tumor effect with the combination of PA. Cryopreservation can increase the immunoactivities of A-LAK cells from the patients with hepatocellular carcinoma. AIM: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and to study the effects of cryopreservation and phenylacetate (PA) on biological characters of A-LAK cells. METHODS: A-LAK cells were obtained from peripheral bloodmo ■onuclear Cells(PBMCs) of the patients with hepatocellular carcinoma(HCC) by using L-phenylalaninemethyl ester(PME)to deplete immunosuppressivemonocytes.Proliferative activity of SMMC7721 cell line aftertreatment with phenylacetate(PA)was observed.A-LAKcells were treated with the supernatant of SMMC7721 Cellsthat had been pretreated with PA.The changes of proliferation,cytotoxicity and phenotype of A-LAK cellswere investigated after cryopreservation.RESULTS:The expansion of A-LAK cells(96.79±69.10 foldson Day 14)was significantly higher than that of non-adherent LAK (NA-LAK) cells (22.77±13.20) as well as conventional LAK cells (4.64±0.91). PA significantlysuppressed the growth of SMMC7721 cells, and the inhibitor ratio was 46%. The supernatant of culture d tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of the supernatantwas previously decreased after treatment with PA.Impairments in proliferation and cytotoxicity of A-LAK cells immediately after thawing of cryopreservation and recoveryafter reincubation with IL-2 were observed .The cytotoxicity of thawed A-LAK cells on Day 5 was significantly higher than that of fresh A-LAK before freezing (54.8±10.2 % vs 40.5±6.4 %).No significant change in the percentage of lymphocyte subsets was identified in frozen A-LAK cells Ascompared with that in the fresh control cells.CONCLUSION: A-LAK cells can be simply prepared by using PME. and showing a synergistic anti-tumor effect with the combination of PA. Cryopreservation can increase the immunoactivities of A-LAK cells from the patients With hepatocellular carcinoma.