Fig. 5

3D-microfluidic system and the NK functionality against PAECs under the flow. (A) Illustration of the cylindrical 3D chip depicting the three spatial coordinates (magenta, yellow, and green) (left). Channel’s length: 1 cm, dimeter: 550 μm. Confocal imaging shows a monolayer of pig endothelial cells (PAECs) covering the inner wall of the channel. Cells were stained with anti-alphaGal antibody (red), and nuclei were stained with Hoechst (cyan). The image was acquired using a Leica confocal SP8 microscope at 20× magnification. Scale bar = 100 μm. (B) The presence of a glycocalyx layer on PAECs in the 3D system under flow conditions without TNF treatment. Cells were stained with anti-heparan sulfate (red), and nuclei were stained with Hoechst (cyan). Images were acquired using a Leica confocal SP8 microscope at 20× magnification. Scale bar = 25 μm. (C) Adhesion molecules on porcine endothelium in 3D static and flow conditions, with and without TNF treatment. At the endpoint, the experimental channels were washed, and the cells were fixed before staining for porcine Vascular adhesion molecule-1 (pVCAM-1) and intercellular adhesion molecule-1 (pICAM-1). Nuclei were stained with Hoechst (cyan). The figure shows the maximum projection of 20 z-stacks with 0.5 μm per stack. Percentage of pICAM-1 and pVCAM-1 positive cells was quantified by dividing the number of positive cells by the total number of nuclei per field. Data represent two independent experiments. Scale bar = 60 μm.