The Efficiency of Density Gradient for Separation of Mesenchymal Stem Cells from Bone Marrow Sample

Background and Objective: Nowadays, mesenchymal stem cells (MSCs) are considered as a promising tool for treatment of different diseases. Due to the low frequency of MSCs, however, it seems inevitable to expand them in vitro prior to use, which could affect the quality of the cells.  In all isolation procedures, the density gradient separation of Ficoll is used for volume reduction and RBC exclusion.  In this study, the efficiency of Ficoll density gradient was evaluated. Materials and Methods: Human bone marrow samples were laid over Ficoll.  Following centrifugation, the upper fraction containing the mononuclear cell layer and the lower fraction (RBC layer) were used for in vitro culture. The number and characteristics of MSCs in both layers were then compared with each other. Results: Inspection of the cultured cells showed that the lower fraction contained MSC-like cells. These cells had spindle–like appearances and exhibited a high capacity for expansion. Furthermore, they showed a potential for differentiating into adipocyte and osteocyte differentiation.  Cytofluorometric analysis showed that these cells were positive for CD۷۳, CD۹۰, and CD۱۰۵, and negative for CD۴۵, CD۳۴, and CD۳۱.  It was also found that this fraction contained ۵۸ ± ۲۲% of the total isolated MSCs.  Conclusion: Density gradient is not a very efficient method for separation of MSCs because it leads to sedimentation of most of the cells to the lower compartment during centrifugation, which results in their exclusion. Therefore, there is a need for developing new methods to obtain larger amounts of MSCs from bone marrow.

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Keywords: Mesenchymal stem cells , Cell separation , Density gradient