Yang Yan-Mei, Li Hong, Zhang Lei, Dang Rui-Jie, Li Ping, Wang Xiao-Yan, Zhu Heng, Guo Xi-Min, Zhang Yi, Liu Yuan-Lin, Mao Ning, Jiang Xiao-Xia, Wen Ning
Department of Stomatology, Chinese PLA General Hospital, Beijing 100853, China.
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Dec;21(6):1563-7. doi: 10.7534/j.issn.1009-2137.2013.06.037.
This study was purposed to establish a convenient and efficient method for isolating and culturing mouse bone marrow mesenchymal stem cells (MSC). The femurs and tibias of mouse were taken under sterile condition. MSC were isolated and cultured with flushing- out bone marrow or collagenase-digested bone fragment or bone marrow plus bone fragment. MSC colony number and size were compared. Immunophenotype and differentiation ability were tested to identify MSC. The results showed that colonies from bone marrow plus bone fragment group came out earliest and the colony number was 20 ± 4 at day 4; there were 11.5 ± 2.5 colonies in collagenase-digested bone fragment group and 9.5 ± 1.5 in flushing- out bone marrow group. The total cell yields of MSC after passaging showed best in bone marrow plus bone fragment group. Flow cytometry data showed the cultured cells expressed Sca-1, CD44 and CD29, not expressed pan-leukocyte surface marker CD45 and endothelial cell marker CD31. The isolated and cultured MSC could differentiate into osteoblast at the osteogenic differentiation condition, or adipocyte at adipogenic differentiation condition. It is concluded that the method of bone marrow plus bone fragment is convenient and efficient for isolating and culturing MSC.
本研究旨在建立一种简便高效的小鼠骨髓间充质干细胞（MSC）分离培养方法。在无菌条件下获取小鼠的股骨和胫骨。采用冲洗骨髓法、胶原酶消化骨碎片法或骨髓加骨碎片法分离培养MSC。比较MSC集落数量和大小。检测免疫表型及分化能力以鉴定MSC。结果显示，骨髓加骨碎片组集落最早出现，第4天集落数为20±4；胶原酶消化骨碎片组有11.5±2.5个集落，冲洗骨髓组有9.5±1.5个集落。传代后MSC的总细胞产量以骨髓加骨碎片组最佳。流式细胞术数据显示，培养的细胞表达Sca-1、CD44和CD29，不表达全白细胞表面标志物CD45和内皮细胞标志物CD31。分离培养的MSC在成骨分化条件下可分化为成骨细胞，在成脂分化条件下可分化为脂肪细胞。结论：骨髓加骨碎片法分离培养MSC简便高效。
