双阴性选择法纯化培养人骨髓多能成体祖细胞 |
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摘 要: |
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关 键 词: | 人骨髓来源多能成体祖细胞 免疫磁珠分离法 分离纯化 细胞培养 流式细胞术 |
In vitro culture of multipotent adult progenitor cells purified from human bone marrow by magnetic-activated cell sorting] |
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Authors: | Ning Mu Yi Gao Li-jun Tang |
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Institution: | Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. mnwsq@163.com |
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Abstract: | OBJECTIVE: To establish a method for purifying and culturing human multipotent adult progenitor cells (MAPCs) in vitro. METHODS: Mononuclear cells were separated from the bone marrow of healthy adult volunteers by density gradient centrifugation and cultivated in adherent culture. The plastic-adherent cultured bone marrow cells were isolated by magnetic-activated cell sorting with CD45 and GlyA magnetic microbeads, and the purity of CD45-/GlyA- cells evaluated by flow cytometry. Phase-contrast microscopy was used to detect morphological changes of the cells in different stages of culture. RESULTS: Approximately (5-10)x10(4)/ml MAPCs could be separated from every 1x10(6)/ml bone marrow mononuclear cells by magnetic- activated cell sorting. The viability of the cells before and after separation was (96.7+/-1.7)% and (96.0+/-2.4)%, respectively. The isolated MAPCs grew well in a self-prepared culture medium till the16th passage. The purity of CD45-/GlyA- separated from the bone marrow was more than 98% as examined by flow cytomety even till the 12th passage. CONCLUSIONS: MAPCs derived from adult human bone marrow can be purified by magnetic-activated cell sorting with CD45 and GlyA microbeads and retain the undifferentiated state for a long time. The self-prepared culture medium is appropriate for MAPCs cultures in vitro. |
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