Regulation of osteogenetic differentiation of mesenchymal stem cells by two axial rotational culture.

It is crucial to understand how gravitational force affects the osteogenic differentiation of mesenchymal stem cells (MSCs), and these fundamental aspects hold promise for the development of a novel model of MSC regulation for cell proliferation and differentiation. The objective of this study was to investigate how significantly gravitational dispersion affects the spontaneously induced osteogenic differentiation of MSCs. Expression of surface antigen was measured by flow cytometry prior to two axial rotational cultures. About 12,500 hMSC cells were spread on culture wells of 1.8 cm(2) surface area and incubated for 7 days at 5% CO(2). The culture medium, 10% FCS/DMEM containing 3 ng/ml bFGF, was replaced every 3 days. Four wells then were placed in a 50-ml centrifugal tube filled with 10% FCS/DMEM without bFGF. The centrifugal tube was attached to the center of the rotor, and two axial rotational cultures were started at 10 rpm each of both rotational speeds. It was confirmed that the hMSCs used in this study expressed typical surface antigens as well as a multipotent differentiation ability for either osteogenic or adipogenic differentiation. Spontaneous expression of alkaline phosphatase (Alp) mRNA following the conventional static culture (1G condition) was suppressed by two axial rotational cultures for 7 days (p < 0.05). A separate study indicated that the cell count number eventually increased from 24,700 ± 6,400 to 78,400 ± 18,700 (p < 0.05). In addition, suppressed Alp mRNA was recovered after an additional 7-day culture under static conditions. This result indicated that dispersion of gravity is a promising modality to regulate osteogenic differentiation of hMSCs.