Supplementary Figure 1: Schemes and characterization of the microfluidic device.

(a) Detail of the two layers for the SU8 mold of the device. In the final device the white parts will be occupied by the PDMS and the colored parts will be empty. (b) Bright-field picture of the gel compartments loaded with colored ink to show how the liquid is retained by the phase-guiding features inside the compartments. (c) Schematic representation of the casting of the PEG-hydrogel (here represented in green) in the gel compartments. (d) Computational simulation of the diffusion of a reference molecule from the source side of the cell chamber. The graphs (right) is the calculated distribution at different time points of a reference molecule at the source edge of the chamber along the blue line reported in the left panel. (e) Computational simulation of the diffusion of a reference molecule from the source side of the cell chamber. The graphs (right) is the calculated distribution at different time points of a reference molecule at the sink edge of the chamber along the blue line reported in the left panel. (f) Schematic representation of the microfluidic device for alternating perfusion. By alternating perfusion through inlet A and inlet B it is possible to temporally control the composition at the source. Inlet C is perfused continuously. (g) Visualization of TexasRed-40kDa Dextran at the specified time points. The device has been perfused intermittently with “medium + TexasRed-40kDa Dextran” through inlet A (6hr), and “medium only” through inlet B (6hr). Inlet C has been perfused continuously with “medium only”. The scheme represents the period of perfusion through inlet A and inlet B. (h) Quantification of the fluorescent signal of TexasRed-40kDa Dextran within the red rectangles reported in “g” for different time-points. Repetition on eight samples showed similar results.