Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.
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.
Carrancio
Soraya
S
Servicio de Hematología, Hospital Universitario de Salamanca, Salamanca, Spain.
López-Holgado
Natalia
N
Sánchez-Guijo
Fermín M
FM
Villarón
Eva
E
Barbado
Victoria
V
Tabera
Soraya
S
Díez-Campelo
María
M
Blanco
Juan
J
San Miguel
Jesús F
JF
Del Cañizo
M Consuelo
MC
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