Experijental Stduy Of Isolation, Culture And Identification Of Goat Bone Marrow Mesenchymal Stem Cells In Vitro

Background and ObjectiveBone marrow mesenchymal stem cells (BMSCs) derived from the mesoderm, mainly exists in the body of the interstitial connective tissue and organs, to a maximum content of the bone marrow. Bone marrow mesenchymal stem cells have self-renewal capacity and multi-differentiation and proliferation potential. Bone marrow mesenchymal stem cells in a specific culture conditions can be differentiated into mesoderm origin of bone cells, stromal cells, cartilage cells, tendon cells and fat cells, bone marrow mesenchymal stem cells can differentiate into endoderm source of hepatic oval cells and ectodermal origin of the glial cells, nerve cells. Bone marrow mesenchymal stem cells because of simple isolation and culture, wide variety of sources, no rejection after transplantation and many other advantages. Bone marrow mesenchymal stem cells in recent years become a tissue engineering cell therapy, gene therapy and organ transplant research focus, in the multi-disciplinary multi-domain as an ideal seed cells have been widely applied. However, bone marrow mesenchymal stem cells, although the source of more widely in the body, but the number of very small, and with the decline in physical function and individual aging, the number will be gradually reduced. Tissue engineering and cell engineering to carry out the first condition is the isolation and cultivation of seed cells. There is now did not find bone marrow mesenchymal stem cells in the direct identification method.The experiment ready to find an ideal method to separate in vitro cultured bone marrow mesenchymal stem cells. Observation of bone marrow-derived mesenchymal stem cell growth characteristics and to explore identification methods. We hope that the experiment can to carry out foundation in tissue engineering and cell engineering.Materials and methodsCells were isolated from healthy male goat iliaca bone marrow by density gradient centrifugation, and cultured with attachment method in vitro cell culture technique. The characteristics of the proliferating and growing BMSCs were observed with phase contrast microscope. The growth curve was drawn to determine doubling time, determination seeding efficiency and survival rate of cells, immunocytochemical method identified the BMSC activity and phenotype.Results1. Primary cell culture morphology:Primary cultured BMSC were oval, spindle-shaped or polygonal, and adhered to plastic surface within 48 hours, cell growth in good condition and 95% cells were alive in passage first to fifth.2. Passaged cell culture morphology:Passage cells showed spindle-shaped under the microscope, swirling radial arrangement at high cell density, the passage cells rate of cell adhesion within 12h above 90%, cell can covered bottom of tissue culture flask in 6-8 days.3. Bone marrow mesenchymal stem cells were identified by Immunocytochemistry: Immunocytochemistry showed the third passage of BMSC were positive for CD29 and CD44,while negative for CD34 and CD45.4. Cell growth curve and doubling time:Cell growth curve growth curve showed S-type, and were in latent phase at 1-3 days, slow cell growth during this period, in logarithmic growth phase 2-3 days later, and growth rate has accelerated noticeably during this period, cells came into platform phase at 6-7 days later. The mean doubling time was (27.44±2.18) hours.Conclusion1. BMSC were successfully isolated from goat bone marrow by density gradient centrifugation and adherent culture method, cell growth in good condition.2. Identification results confirm that the cultured cells is goat bone marrow mesenchymal stem cells(BMSC).