In vitro purification and culture of multipotent adult progenitor cells from human bone marrow by magnetic activated cell sorting / 第二军医大学学报

ABSTRACT

Objective: To establish a method for isolation, purification, and culture of human multipotent adult progenitor cells (MAPCs) in vitro, so as to lay a foundation for the application of hMAPCs in tissue-engineering research and clinical medicine. Methods: The mononuclear cells were obtained from bone marrow of healthy volunteers by gradient centrifugation and were subjected to adherence culture. The adherent cells were then subjected to magnetic activated cell sorting (MACS) (depletion selection with CD45 and GlyA microbeads). Then the purity of selected cells was identified by flow cytometer. The growth of the purified cells was observed and the expression of CD29, CD44, CD34, and HLA-DR was analyzed by flow cytometers. Results: (1) Averagely (5-10) × 104/ml hMAPCs could be separated from 1 × 106/ml bone marrow mononuclear cells by MACS. The cell viability was similar before ([96.7±1.7]%) and after ([96.0±2.4]%) isolation. (2) The isolated MAPCs grew well in the self-designed culture medium and could be passaged for 20 generation. (3) The purity of the CD45- and GlyA- cells separated from bone marrow adherent cells was more than 98% as determined by flow cytometer. (4) In hMAPCs, the positive rate was 99.2% for CD29, 98.3% for CD44, 1.2% for CD34, and 5.3% for HLA-DR. Conclusion: (1) The bone marrow-derived hMAPCs can be purified by MACS through depletion selection of CD45 and GlyA microbeads. (2) The hMAPCs can be expanded in vitro in self-designed medium, maintaining a non-differentiation state for a long time.