Figure 2. Effect of conditioned medium on adipogenic differentiation.
ASCs were loaded at different cell suspension densities, 0.5×105, 2×105 or 4×105 cells/mL, and induced to differentiate at a flow rate of 500 nL/min in AM or CM at two different concentrations of serum and adipogenic factors. CM was a 1∶1 mixture of supernatant from ASCs undergoing differentiation in batch cultures and fresh AM to ensure sufficient supply of nutrients. 1.5×AM was used in the mixture instead of 1×AM to compensate for an expected consumption/degradation of the adipogenic stimuli in the collected supernatant. A) Relative lipid accumulation in relative units. Lipid accumulation in the chambers are taken as a measurement of extent of differentiation, and was determined by quantifying the total area of lipid-filled droplets in each chamber (within the rectangle indicated by the dotted line in figure 2c) divided by the corresponding total cell area at the start of differentiation, see also material and methods. I and II denote two independent experiments. AM (low conc.) and CM (AM low conc.) indicate AM with 4 times lower concentrations of serum and adipogenic inducers and CM based on the 4 times lower concentrated AM. B) 10x phase contrast images 14 days after differentiation initiation of a representative area of a chamber at the different tested conditions. C) Scan of an entire cell culture chamber. The rectangle indicated by the dotted line shows the area of a cell culture chamber used for measurements of extent of differentiation with respect to total lipid area, lipid vacuole area per cell and fraction of differentiated cells (Figure 3 and Figure S3).