Modified whole bone marrow adherent method for the isolation and purification of rats bone marrow mesenchymal stem cells

Abstract

Objective To explore an effective method for the isolation and purification of rats bone marrow mesenchymal stem cells (BMSCs) and to study the basic biological characteristics of BMSCs. Methods One healthy Sprague-Dawley (SD) rat of specific pathogen free (SPF) grade aged 4 weeks was selected, and the BMSCs were isolated using the modified whole bone marrow adherent method. The morphologic variation and proliferation features of BMSCs were observed under inverted phase contrast microscope, and the surface markers of BMSCs of P3 generation were detected by flow cytometry. Cell growth curves were drawn using the cell counting kit (CCK)-8 assay. Results Typical adherent growth was observed in the primary BMSCs with the forms of fusiform or polygon and appearance of circinate cell colony. The cell colonies fused with each other up to 80% in about 8 d. The purified passage cells still kept a homogeneous morphology and a high potential of proliferation. The growth curve of BMSCs indicated an S shape. The surface markers of BMSCs of P3 generation cluster of differentiation (CD) 29 and CD90 were observed positive, but CD11b and CD45 negative. Conclusions BMSCs with high-purity, active proliferation and stable biological characteristics can be cultured using modified whole bone marrow adherent method. The growth curve of BMSCs presents in an S shape. Key words: Mesenchymal stem cells; Whole bone marrow adherent method; Rats