title: Effects of cryopreservation and phenylacetate on biological characters of adherent LAK cells from patients with hepatocellular carcinoma.
abstract: OBJECTIVE
  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 blood mononuclear cells (PBMCs) of the patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. Proliferative activity of SMMC7721 cell line after treatment with phenylacetate (PA) was observed. A-LAK cells were treated with the supernatant of SMMC7721 cells that had been pretreated with PA. The changes of proliferation, cytotoxicity and phenotype of A-LAK cells were investigated after cryopreservation.
  
  
  RESULTS
  The expansion of A-LAK cells (96.79 +/- 69.10 folds on 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 significantly suppressed the growth of SMMC7721 cells, and the inhibitor ratio was 46%. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of the supernatant was previously decreased after treatment with PA. Impairments in proliferation and cytotoxicity of A-LAK cells immediately after thawing of cryopreservation and recovery after 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 as compared with that in the fresh control cells.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China. slye@shmu.edu.cn
authors.forename: Ning
authors.initials: N
authors.lastname: Zheng
authors.forename: Sheng-Long
authors.initials: SL
authors.lastname: Ye
authors.forename: Rui-Xia
authors.initials: RX
authors.lastname: Sun
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Zhao-You
authors.initials: ZY
authors.lastname: Tang
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000276:immunology*; D002448:Cell Adhesion; D015925:Cryopreservation; D003601:Cytotoxicity Tests, Immunologic; D006801:Humans; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology*; D008113:Liver Neoplasms / Q000276:immunology*; D016131:Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism*; D010648:Phenylacetates / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D010648:Phenylacetates; C025136:phenylacetic acid