Study Of The Isolation Culture, Identification And Biological Characteristics Of Rabbit Bone Marrow Mesenchymal Stem Cells

Objective: To establish a method for the isolation culture , multiplication and identification of marrow mesenchymal stem cells from rabbits in vitro, observe their biological features and supply fundament theory for tissue engineering .Methods: MSCs was harvested from crista iliaca. Bone marrow aspirate of New Zealand white rabbit was density gradient centrifuged and cultured in plastic culture bottles. The cells were examined by invert microscope and transmission electron microscope.The growth curve was drawn and cell doubling time, confluent rate, cell activity was measured. MSCs quality was evaluated with immunocytochemistry ,and their phenotypical properties were known.Results: MSCs had soindle shape and a typical morphological fibroblast.The primary cells were confluent in single layer after being plated for 12-15 days. The cells were noted to have a great expansive potential after subculture. The average time for cell doubling was 34 hours.The proliferation ability declined with time of the culture. About 90% subcultured cells dahered the wall in 12h in passage 1, 3 and 5. 95% cells were alive in passage 1 to 5, decreased from 6 passage. The cells showed rapid proliferation ablity in electron micoscope. MSCs uniformly expression of CD₄₄ and did no expression of CD34.Conclusion: The combination of density gradient centrifugation andadere culture is an effective method to isolate MSCs from bone marrow. MSCs cultured in vitro show stable growth , rapid proliferation and 1 to 5 passage can be used as seed cells in cartilage tissue engineering. The surface antigens,which are positive for CD44 and negative for CD34 ,are a method for identification of MSCs.Postgraduate student: Yu-quan Dong(orthopedics) Directed by Prof.: Xue-ren Teng...