title: [Influence of Cryopreservation on Human Peripheral Blood Mononuclear Cell Immunocompetence].
abstract: OBJECTIVE
  To establish a method for isolation, cryopreservation and recovery of the highly viable human peripheral blood monomuclear cells (PBMNCs) so as to achieve the long-term preservation of PBMNCs.
  
  
  METHODS
  A total of 80-100 ml peripheral blood were collected from the healthy volumteers aged over 50 years old. The PBMNCs were isolated by the Ficoll density gradient technique and cryopreserved gradually by program control method in liquid nitrogen freezer of -196 °C. The serum-free medium and autoloqous plasma medium were test for preservation of PBMNCs. The cell viability was assessed at time point of 1, 2, 4, 8, 12 and 24 months after thawing. Finally, the proliferation ability, purity and cytotoxicity were compared between the autologous immune lymphocytes (AIL) induced from cryopreserved PBMNCs and AIL as control from fresh PBMNCs.
  
  
  RESULTS
  After separating, the cell viability was 99.6%±0.4%, and the recovery rate of lymphocytes was 58.4%±6.52%. The cell recovery rate of lymphocyte was 89.7%±3.82% at 24 months. The quality assurance program was reliable within 2 years of running. The AIL cells induced with cryopreserved PBMNCs were not significantly different from those induced from fresh PBMNCs in terms of proliferative action, purity and cytotoxicity(CD3(+)CD8(+) ≥45%,CD3(+)CD56(+) NKT≥10%,CD4(+)CD25(+) NKT≤10%).
  
  
  CONCLUSIONS
  Manual separation of lymphocytes in vitro can get enough high-quality PBMNCs. The long-term cryopreserved PBMNC still maintain their high viability. The reinfusion of the clinical autologous immune cells would be advantageous for early tumor immunotherapy. Human AIL induced from cryopreserved PBMNC maintain their anti-tumor ability. These findings have the important implications for the application of these cells to adoptive cellular therapy.
authors.affiliation: Department of Obstetrics and Gynecology, The First Hospital of Jilin University, Changchun 130000, Jilin Province, China.
authors.forename: Xue-Feng
authors.initials: XF
authors.lastname: Pan
authors.affiliation: Jilin Yinfeng Biological Engineering Technology Co Ltd, Changchun 130000, Jilin Province, China.
authors.forename: Chun-Xia
authors.initials: CX
authors.lastname: Lu
authors.affiliation: Department of Obstetrics and Gynecology, The First Hospital of Jilin University, Changchun 130000, Jilin Province, China.
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Yang
authors.affiliation: Department of Obstetrics and Gynecology, The First Hospital of Jilin University, Changchun 130000, Jilin Province, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Shu
authors.affiliation: Jilin Yinfeng Biological Engineering Technology Co Ltd, Changchun 130000, Jilin Province, China;
authors.forename: Na
authors.initials: N
authors.lastname: Yao
authors.affiliation: Jilin Yinfeng Biological Engineering Technology Co Ltd, Changchun 130000, Jilin Province, China;
authors.forename: Hong-Bin
authors.initials: HB
authors.lastname: Zuo
authors.affiliation: Department of Obstetrics and Gynecology, The First Hospital of Jilin University, Changchun 130000, Jilin Province, China.E-mail：cuilifeng@163.com.
authors.forename: Li-Feng
authors.initials: LF
authors.lastname: Cui
mesh_terms: D002470:Cell Survival; D015925:Cryopreservation; D006801:Humans; D007121:Immunocompetence; D007963:Leukocytes, Mononuclear