Characterization of mouse clonal mesenchymal stem cell lines established by subfractionation culturing method

Figure 1 Flow diagram of the “subfractionation culturing method” used to establish mouse clonal mesenchymal stem cell lines. Mouse bone marrow aspirate was mixed with Dulbecco’s modified Eagle’s Medium-low glucose and plated onto a 100-mm cell culture dish. After 2-h incubation, the supernatant only was transferred to a new dish. This procedure was repeated two more times, and then the supernatant was subsequently transferred to cell culture dishes with a 1- or 2-d interval as shown. Each dish was then incubated until single-cell-derived colonies appeared. When colonies of cells were large enough, they were transferred to a six-well plate or 100-mm cell culture dish and then expanded to larger flasks for freezing and further study. Unique mouse clonal mesenchymal stem cell (mcMSC) lines were saved in a mcMSC library.