Quantitative analysis of cytokines, chemokines, growth factors and other soluble proteins in different biological liquids is routinely performed in contemporary diagnostics and biomedicine research. However, current methods of analysis are time-consuming and include multiple steps. In this study, we have developed a protocol that describes how bio-layer interferometry can be applied to quantify an analyte in several minutes. Conditioned growth media collected from mouse mesenchymal stem cells grown in normoxia or hypoxic conditions in a monolayer fashion, MSC-derived 3D cell sheets and 3D spheroids were used as a model system in which we determined a concentration of vascular endothelial growth factor (VEGF-A). This technique displayed a high sensitivity (down to 0.1 ng of VEGF-A per mL as a minimum). The measured concentrations of VEGF-A in the conditioned media from mesenchymal stem cells turned out to be similar with values determined by the enzyme-linked immunosorbent assay. Using bio-layer interferometry, it was shown that as compared to mesenchymal stem cells grown in monolayer, spheroids and 3D sheets of mesenchymal stem cells produce significantly more VEGF-A (by 2.5-3.0-fold). Thus, due to the developed protocol it was possible to adapt bio-layer interferometry for rapid quantification of growth factors in conditioned media.
Characteristic differences of cell sheets composed of mesenchymal stem cells with different tissue origins
