2022, Vol. 26 ›› Issue (24): 3814-3820

A new method for extracting human bone marrow mesenchymal stem cells and the comparison with traditional methods

Li Shidan1, Xing Wei2, Xie Xiaoyu1, Li Youbin1, Wang Shaochuan1, Fei Jun3   

Abstract: BACKGROUND: The methods to extract human bone marrow mesenchymal stem cells, including whole bone marrow adherent culture, density gradient centrifugation, flow cytometry separation and immunomagnetic bead separation. Flow cytometry separation and magnetic bead separation are restricted on equipment and reagents, so they are limited to applied. The whole bone marrow adherent culture method and density gradient centrifugation method are commonly used; however, there are still some shortcomings, such as slow cell growth rate and long centrifugation time. Thus, the efficiency of the experiment will be further improved if a more convenient and short time extraction method of bone marrow mesenchymal stem cells is found.
OBJECTIVE: To introduce a new method of extracting human bone marrow mesenchymal stem cells and compare the extracted cells with those extracted by traditional methods to prove the feasibility and practicability of the new method.  
METHODS: Phosphate buffered saline was mixed with human bone marrow tissue at the same ratio, and the mixture was stratified by centrifugation. Human bone marrow mesenchymal stem cells were extracted from the white membrane layer followed by comparison with the cells extracted using whole bone marrow adherent method. The extracted cells were identified as bone marrow mesenchymal stem cells by flow cytometry and multidirectional differentiation potential. The number of primary cells, cell morphology, size, cytoskeleton, expression of surface markers, multidirectional differentiation potential, colony formation, proliferation, migration, and osteogenic differentiation were analyzed by cell counting, crystal violet staining, cytoskeleton staining, scratch test, Transwell migration test, and RT-PCR.
RESULTS AND CONCLUSION: (1) After centrifugation, human bone marrow samples were stratified using phosphate buffered saline to extract human bone marrow mesenchymal stem cells. The number of primary cells extracted by phosphate buffered saline method was more than that of whole bone marrow adherent method. The floating adipose tissue was less in the culture system, and the primary cells grew faster. (2) The cells extracted by phosphate buffered saline and whole bone marrow adhesion were fusiform and expressed CD90, CD105, and CD73, but did not express CD45, CD34, CD11b, CD19, and HLA-DR. They had multidirectional differentiation potential and met the standard of mesenchymal stem cells. (3) There was no significant difference in morphology, size and cytoskeleton of the cells extracted using the two methods. Both groups of cells could form colonies and had migration ability. After osteogenic differentiation, both groups of cells expressed osteoblast markers. (4) The results show that it is feasible and practical to extract human bone marrow mesenchymal stem cells with phosphate buffered saline under certain conditions.
Key words: mesenchymal stem cells, bone marrow mesenchymal stem cells, phosphate buffered saline, whole bone marrow adhesion, cell extraction, cell identification