title: Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.
abstract: OBJECTIVE
  Optimization of the mesenchymal stem cells (MSC) isolation and expansion method.
  
  
  METHODS
  Mononuclear cells (MNC) from bone marrow aspirates were obtained by both density gradient centrifugation (standard method) and gravity sedimentation. Cells were cultured in standard conditions (10% fetal calf serum and normal oxygen tension [21% O(2)]) and expansion results compared to those obtained with the same culture conditions to which platelet lysate (PL) preparations were added; in addition, the 21% O(2) concentration was compared to a lower (5%) concentration (hypoxia) until the fourth cell passage. Time of expansion, number of cells obtained, morphology, cell surface markers, and differentiation potential were evaluated.
  
  
  RESULTS
  MSC obtained by any of the different culture conditions expressed comparable immunophenotype and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. When the number of MSC obtained at fourth passage was analyzed, the highest cell numbers were obtained with gravity sedimentation isolation and PL-supplemented culture and the expansion time was the shortest when cells were cultured under hypoxic conditions.
  
  
  CONCLUSIONS
  MSC isolation by MNC gravity sedimentation together with culture medium supplementation with 5% of PL in a hypoxic atmosphere (5% O(2)) significantly improved MSC yield and reduced expansion time compared to the standard accepted protocols.
authors.affiliation: Servicio de Hematología, Hospital Universitario de Salamanca, Salamanca, Spain.
authors.forename: Soraya
authors.initials: S
authors.lastname: Carrancio
authors.forename: Natalia
authors.initials: N
authors.lastname: López-Holgado
authors.forename: Fermín M
authors.initials: FM
authors.lastname: Sánchez-Guijo
authors.forename: Eva
authors.initials: E
authors.lastname: Villarón
authors.forename: Victoria
authors.initials: V
authors.lastname: Barbado
authors.forename: Soraya
authors.initials: S
authors.lastname: Tabera
authors.forename: María
authors.initials: M
authors.lastname: Díez-Campelo
authors.forename: Juan
authors.initials: J
authors.lastname: Blanco
authors.forename: Jesús F
authors.initials: JF
authors.lastname: San Miguel
authors.forename: M Consuelo
authors.initials: MC
authors.lastname: Del Cañizo
mesh_terms: D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D005260:Female; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D009000:Monocytes / Q000166:cytology