双阴性选择法纯化培养人骨髓多能成体祖细胞

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (05): 535-537.

双阴性选择法纯化培养人骨髓多能成体祖细胞

慕宁, 高毅, 唐力军

南方医科大学珠江医院普外科, 广东, 广州, 510282

出版日期:2005-05-20 发布日期:2005-05-20
基金资助:
收稿日期:2004-10-20。
基金项目:广东省“十五”重点科技攻关专项基金资助项目(2002A3020206)
作者简介:慕宁(1976- ),男,在读硕士研究生,电话:020-61643210,E-mail:mnwsq@163.com

In vitro culture of multipotent adult progenitor cells purified from human bone marrow by magnetic-activated cell sorting

MU Ning, GAO Yi, TANG Li-jun

南方医科大学珠江医院普外科, 广东, 广州, 510282

Online:2005-05-20 Published:2005-05-20

摘要/Abstract

摘要： 目的建立体外分选纯化及培养人骨髓来源多能成体祖细胞(MAPCs)的方法.方法取健康成人志愿者适量骨髓采用梯度密度离心法分离获取单个核细胞,贴壁培养后,将获取的骨髓贴壁细胞通过CD45、血糖蛋白A(GlyA)免疫微磁珠(miniMACS)负分选,流式细胞术鉴定分选后细胞纯度;对传代到不同阶段的细胞进行CD45、GlyA流式细胞分析,初步了解MAPCs在培养、增殖过程中的免疫学稳定性.结果通过miniMACS分选,平均每1×10⁶/ml骨髓贴壁细胞可分选出约(5～10)×10⁴/ml的MAPCs,分选前后细胞活力分别为(96.7±1.7)%和(96.0±2.4)%,无明显差异;自制的培养基培养分选后的MAPCs生长良好,最长传代到第16代;流式细胞仪分析获取的CD45、G1yA-细胞纯度大于98%;流式细胞仪分析传代第4、8、12代MAPCs,CD45^-、G1yA^-细胞纯度大于98%.结论 CD45、GlyA miniMACS负分选可从骨髓中分离高纯度的MAPCs;MAPCs在本室自制的人MAPCs培养基中体外有较强的增殖能力,且能保持较长时间的稳定状态.

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)×10⁴/ml MAPCs could be separated from every 1×10⁶/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-cells 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.

中图分类号:

R318.14

慕宁, 高毅, 唐力军. 双阴性选择法纯化培养人骨髓多能成体祖细胞[J]. 南方医科大学学报, 2005, 25(05): 535-537.

MU Ning, GAO Yi, TANG Li-jun. In vitro culture of multipotent adult progenitor cells purified from human bone marrow by magnetic-activated cell sorting[J]. Journal of Southern Medical University, 2005, 25(05): 535-537.

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