title: [The induction and cryopreservation of erythroid progenitor cells derived from umbilical cord blood mononuclear cells].
abstract: OBJECTIVE
  To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).
  
  
  METHODS
  UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.
  
  
  RESULTS
  With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.
  
  
  CONCLUSIONS
  This non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.
authors.affiliation: South China Research Center for Stem Cell & Regenerative Medicine; The Lab of Stem Cell and Regenerative Medicine, Beijing Institute of Transfusion Medicine, AMMS, Beijing 100850, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Xie
authors.forename: Jiafei
authors.initials: J
authors.lastname: Xi
authors.forename: Yang
authors.initials: Y
authors.lastname: Lyu
authors.forename: Yu
authors.initials: Y
authors.lastname: Tian
authors.forename: Daqing
authors.initials: D
authors.lastname: Liu
authors.forename: Wen
authors.initials: W
authors.lastname: Yue
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Li
authors.forename: Xue
authors.initials: X
authors.lastname: Nan
authors.forename: Siting
authors.initials: S
authors.lastname: Li
authors.forename: Zeng
authors.initials: Z
authors.lastname: Fan
authors.forename: Xuetao
authors.initials: X
authors.lastname: Pei
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D004900:Erythroblasts / Q000166:cytology; D015672:Erythroid Precursor Cells / Q000166:cytology*; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D014470:Umbilical Cord