Isolation And Identification Of Rabbit Bone Marrow Mesenchymal Stem Cells By Natural Sedimentation Velocity Method

BACKGROUND:In previous studies, it was satisfaction to sorting bone marrow mesenchymal stem cells based on natural subsidence cell with hypotonic. The study based on particle free settling theory, derived the settling velocity of bone marrow mesenchymal stem cells in culture liquid. Establish a simple and easy method of separation and purification of bone marrow mesenchymal stem cells by natural sedimentation velocity.OBJECTIVE:Isolate and identify rabbit bone marrow mesenchymal stem cells preliminary by natural sedimentation velocity method, observe its the value-added potential, cell purity, cell differentiation.METHODS:The density interval of bone marrow mesenchymal stem cells (ρ1) was determined by density gradient centrifuge. The diameter of bone marrow mesenchymal stem cells (d) was measured by electron microscopic. The density of culture liquid (ρ2) was measured by liquid density meter. The viscosity of the culture liquid(μ) was measured by viscosity meter. The settling velocity of BMSCs(Vt) was derived from the above four numerical with the appropriate formula. BMSCs were isolated from bone marrow tissue by natural sedimentation velocity method. BMSCs were iduced into osteoblasts and adipocytes. Cell morphology was observed and alkaline phosphatase (AKP), Von Kossa, oil red O detection were performed. The primary culture time of three different cell sorting method was recorded. The colony formation rate of rabbit bone marrow mesenchymal stem cell of primary cultured was determinated. The results of primary culture time and colony formation rate were analysed by statistics.RESULTS:1. BMSCs seperated by density gradient centrifugation adhered to the plastic, displaying long fusiform shape and shoal arrangement.21 days after osteogenesis or adipogenesis inducing, the third passage cells could be found black calcium nodules through Von Kossa staining, hyacinthine alkaline phosphatase or brown particle through AKP staining, light blue nucleus or orange lipid droplet, which was negative in the control group. The diameter of rabbit BMSCs was measured as 20.37±4.58μm(17μm-28μm) under the electron microscope, and the velocity of rabbit BMSCs in DMEM was 50-55mm/h.2. As by density gradient centrifugation, BMSCs seperated by natural sedimentation velocity adhered to the plastic, showing long fusiform shape and shoal arrangement.3. Compared with the density gradient centrifugation group, the natural sedimentation velocity showing higher level of proliferative activity according to the cell growth curve analysis. Morever, the time in primary stage was 14.93±1.20 days in 1.067g/ml-group,14.89±0.91 days in 1.070g/ml-group, whereas 7.64±0.82 days in natural sedimentation velocity group. The colony formation rate in three groups was different(1.067g/ml-group vs. 1.070g/ml-group vs. natural sedimentation velocity group:5.33±1.07 vs. 5.56±0.88 vs.6.11±1.17,P=0.000).4. Similar to the density gradient centrifugation group, the BMSCs isolated by natural sedimentation velocity was positive in experiments of Von Kossa, AKP or Red oil staining after osteogenesis or adipogenesis inducing, which was negative without inducing.CONCLUSION:1. The diameter of rabbit BMSCs was (20.37±4.58) μm, the velocity of rabbit BMSCs was 50-55mm/h.2. BMSCs can be successfully isolated from bone marrow tissue of rabbit by this method, which can be iduced into osteoblasts and adipocytes.3. The natural sedimentation velocity cost less time than the other two groups of density gradient centrifugation in the primary cultured stage. The colony formation rate of settling velocity group is higher than other two groups.4. Natural sedimentation velocity does not impose any intervention measures for sorting cells, which may be maximum maintaining cell viability and biological characteristics. The whole separation process is simple and safety, which may be little damage to the cells.