NMRI جداسازي و تكثير سلول هاي بنيادي مزانشيمي از مغز استخوان موش

Isolation and in vitro Proliferation of Mesenchymal stem cells from NMRI Mouse Bone Marrow

Objective: The aim of this study was to isolate mesenchymal stem cells from bone marrow NMRI Mice of and determine their growth characteristics in vitro. Materials and Methods: 4-8-week old NMRI mice were killed by cervical dislocation and their tibia and femur were dissected and cleaned from the soft tissues. Bone marrow was then flushed out of the bone and primary cultures were none. One week after culture, the first passage was performed and the cells were seeded with a very low density on wells of 6-well plate. The culture was maintained until a population of fibroblastic cells emerged. At this point the next passages were carried out and the cells were expanded by four passages and frozen for further examination. To prove the mesenchymal nature of the isolated cells, differentiation into bone and adipocyte were carried out. To determine the best fetal bovine serum concentration and initial cell density supporting the maximum cell proliferation, different concentration of (fetal calf serum) FCS and cell density were evaluated the colonogenic potential of the isolated cells were tested by colony forming unit- fibroblast assay (CFU-F). Results: The cells in primary culture were morphologically heterogeneous ranging from round cells to star and spindle- shaped cells. Two weeks after the first passage a population of fibroblastic cells with high proliferation potential dominated the culture These cells readily differentiated to osteoblasts and adipocytes. The isolated cells had a maximum proliferation in a medium with 15% FCS and initiation of culture with I00 cell/cm2 resulted in high-fold increase in cell number. Mesenchymal stem cells isolated in this study, showed high colonogenic activity. Conclusion: MesenchymaI stem cells from NMRI mice can be isolated by low density culture conditions. These cells may have maximum proliferation when cultured with 100cell/cm2 in a medium with 15% FCS.