Figure 2

Design of the microfluidic device reconstructing the 3D immune-cancer spaces to track DC-cancer cells dialogue. (a) Schematic model of the 3D microfluidic device used for real time monitoring of DC migration toward cancer cells and their interactions. The device is composed of a 1.2 mm-wide central immune-chamber, with rounded loading reservoirs at both ends for DC loading, connected through a network of narrow microgrooves (connecting-channels) (10 × 12 × 200 µm, H × W × L) to two side tumor-chambers (150 × 500 × 1000 µm) end-closed with rounded loading reservoirs and designed to recreate 3D tumor spaces. Two external medium chambers ensure the optimal gas and nutrient exchange to the cell cultures. An enlarged 3D section of the device is shown. The boxed area represents a magnification of the connecting-channels. (b) Fluorescence image of the immune-tumor area of the device analysed in microfluidic experiments (left panel) showing the immune-chamber filled with PKH67 green-stained DCs, the connecting-channels and the tumor-chamber loaded with type I collagen embedded-PKH26 red-stained cancer cells. Images were acquired with an inverted Olympus IX73 microscope using a Plan Achromat 4×/0.10NA objective and processed with ImageJ software. Scale bar 100 μm. The inset photographs of the 3D projections represent: (1) a 3D rendering of a confocal image of SW620 cancer cells in the reconstructed 3D-matrix tumor space. Scale bar, 100 μm; (2) a 3D rendering of a confocal image of the connecting-channels entering the 3D-matrix tumor space showing IFN-DCs moving toward cancer cells; scale bar, 100 μm. Image stacks (100 images of 1 μm Z-step size) were acquired using a spinning disk microscope with a 20×/0.45NA objective and processed with Imaris software; (3) brightfield and green fluorescence merged image (20×) of a representative connecting-channel crossed by one IFN-DC actively moving toward the 3D-matrix tumor space. The image was processed with ImageJ software. (c) Schematic representation of isometric views of microfluidic devices showing the two experimental configurations used: no-competition and competition settings. (d) A 3D graphic representation of a section of the microfluidic device showing the interconnection between immune and tumor spaces and reproducing the motion of migrating DCs from the immune-chamber, crossing connecting-channels, toward the tumor-chamber, where cancer cells are targeted by infiltrated DCs.