OBJECTIVE: To explore a suitable method for isolation, purification and multiplication of bone marrow mesenchymal stem cells (BMSCs). METHODS: Density gradient centrifugation and adherence separation methods were applied for isolation of BMSCs from Wistar rats. The cells were cultured and proliferated in culture medium containing calf serum (CS), fetal bovine serum (FBS), free of serum or different volume fraction of FBS. The characteristic and the morphology of BMSCs were observed under inverted microscope every day. The growth curves were draw and the surface antigen of BMSCs were detected by immunocytochemistry technique. The microstructure was observed by transmission electron microscope (TEM). RESULTS: The pure primary cells can be procured by density gradient centrifugation. But the primary cells cultured by adherence separation methods demonstrated higher cytoactive, more rapid proliferation, earlier colony confluence and shorter time for passage than that cultured by density gradient centrifugation method. The cells by adherence separation methods were essentially purified at passage 4. Both CS and FBS can promote the growth and proliferation of BMSCs, but the colony forming efficacy of cells (46.50%) cultured in medium containing 0.12 volume fraction FBS was the highest. The cells surface markers CD44, CD90 were positive and CD14, CD45 were negative. BMSCs were observed by TEM and possessed the characteristic of stem cells. Conclusion BMSCs with high quality and activity can be obtained with adherence separation by suitable method and culture conditions. L-DMEM medium containing 0.12 volume fraction FBS showed more profitable for the growth and proliferation of BMSCs.
OBJECTIVE: To explore a suitable method for isolation, purification and multiplication of bone marrow mesenchymal stem cells (BMSCs). METHODS: Density gradient centrifugation and adherence separation methods were applied for isolation of BMSCs from Wistar rats. The cells were cultured and proliferated in culture medium containing calf serum (CS), fetal bovine serum (FBS), free of serum or different volume fraction of FBS. The characteristic and the morphology of BMSCs were observed under inverted microscope every day. The growth curves were draw and the surface antigen of BMSCs were detected by immunocytochemistry technique. The microstructure was observed by transmission electron microscope (TEM). RESULTS: The pure primary cells can be procured by density gradient centrifugation. But the primary cells cultured by adherence separation methods demonstrated higher cytoactive, more rapid proliferation, earlier colony confluence and shorter time for passage than that cultured by density gradient centrifugation method. The cells by adherence separation methods were essentially purified at passage 4. Both CS and FBS can promote the growth and proliferation of BMSCs, but the colony forming efficacy of cells (46.50%) cultured in medium containing 0.12 volume fraction FBS was the highest. The cells surface markers CD44, CD90 were positive and CD14, CD45 were negative. BMSCs were observed by TEM and possessed the characteristic of stem cells. Conclusion BMSCs with high quality and activity can be obtained with adherence separation by suitable method and culture conditions. L-DMEM medium containing 0.12 volume fraction FBS showed more profitable for the growth and proliferation of BMSCs.
Authors: Eduardo Mansilla; Vanina Díaz Aquino; Daniel Zambón; Gustavo Horacio Marin; Karina Mártire; Gustavo Roque; Thomas Ichim; Neil H Riordan; Amit Patel; Flavio Sturla; Gustavo Larsen; Rubén Spretz; Luis Núñez; Carlos Soratti; Ricardo Ibar; Michiel van Leeuwen; José María Tau; Hugo Drago; Alberto Maceira Journal: Stem Cells Int Date: 2011-06-13 Impact factor: 5.443