title: Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23-25 years.
abstract: OBJECTIVE
  To evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs).
  
  
  METHODS
  Samples of human BMCs that were cryopreserved for 23-25 years (n=20) were thawed to obtain an initial culture and a primary culture (P(0)) that was propagated through five passages (P(1)-P(5)) to obtain MSCs. Freshly collected human bone marrow samples (n=20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P(3) cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing.
  
  
  RESULTS
  In the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p<0.05) while the relative rate of recovery of MSCs was only 48.5±8.6% in P(0). At the end of P(3), fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p>0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P(3) from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells.
  
  
  CONCLUSIONS
  Using the methods described here, long-term (23-25 years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.
authors.affiliation: Department of Haematology, PLA Navy General Hospital, Beijing 100048, China. shenjianliang@csco.org.cn
authors.forename: Jian-liang
authors.initials: JL
authors.lastname: Shen
authors.forename: You-zhang
authors.initials: YZ
authors.lastname: Huang
authors.forename: Shi-xia
authors.initials: SX
authors.lastname: Xu
authors.forename: Pei-hao
authors.initials: PH
authors.lastname: Zheng
authors.forename: Wen-jie
authors.initials: WJ
authors.lastname: Yin
authors.forename: Jian
authors.initials: J
authors.lastname: Cen
authors.forename: Li-zhong
authors.initials: LZ
authors.lastname: Gong
mesh_terms: D017667:Adipocytes / Q000166:cytology; D015703:Antigens, CD / Q000032:analysis; D018070:Biological Specimen Banks / Q000706:statistics & numerical data*; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D002452:Cell Count; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D009474:Neurons / Q000166:cytology; D010006:Osteoblasts / Q000166:cytology; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide