Quantification of chondrogenic differentiation in monolayer cultures of mesenchymal stromal cells

Determining inhibitory or supportive effects of biological, chemical or physical factors on cell fates, including chondrogenic differentiation of mesenchymal stromal cells (MSCs), requires quantification techniques that are rapid, reproducible, and able to monitor these effects over time. Methods currently used to analyze chondrogenic differentiation are either qualitative staining procedures or indirect DNA quantifications. Because of these limitations, further methods are needed to improve determination of chondrogenic differentiation. In the present study, we applied a histological staining method, which is established for investigation of articular cartilage degeneration by use of Safranin O dye, on chondrogenic differentiated cells in monolayer cultures. MSCs were differentiated on 12-well formats, at increasing concentrations of TGF-β3, cell numbers, and incubation times. Quantification was performed by solubilizing the adsorbed dye into isopropanol followed by determining the optical density (O.D.) through spectrophotometry. Our results show that the O.D. is directly related to cell numbers and incubation periods, and that the technique is applicable to study agents which affect chondrogenic differentiation.