Separation, culture and identification of rabbit bone marrow mesenchymal stem cells by iliac puncture:operation details and techniques

Zhang Cong; Liu Hong-mei; Li Qing-wei; Chen Guo-wu; Liang Xiao; Meng Chun-yang

doi:10.3969/j.issn.2095-4344.2014.23.006

Abstract：BACKGROUND:Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner. OBJECTIVE:To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation. METHODS:Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cellmorphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cellphenotype identified by flow cytometry. RESULTS AND CONCLUSION:We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After careful y summarizing and analysis of the reasons, the operation was successful y completed on the rest 11 rabbits. Bacteria pol ution and cellaging were not found in the process of cellculture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cellgrowth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure wil be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stem cells, which lays a good foundation for celland animal experiments in the future.