Live EV exchange on the microfluidic chip: To verify the ability of our chips to serve as reliable platforms for live EV exchange, we monitored transmigration of secreted EVs across the Matrigel channel and their internalization by cells in the recipient channel. (a) A Matrigel-loaded chip was injected in the donor channel with U937-XP cells, and the cells were visualized 1 h later using a GFP emission filter (original magnification = 1000×; 3D deconvoluted.). (b) Matrigel-loaded chips were injected into the donor channel with either U937-XP cells or U937 cells as a negative control, and the Matrigel-loaded matrix channel was visualized at 1 h, 24 h, 48 h, and 72 h for the presence of fluorescence from diffusing vesicles using a GFP emission filter (Matrigel channel ribs are outlined with dashed lines. Original magnification = 1000×). (c) A Matrigel-loaded chip with U937-XP cells injected into the donor channel, and with an empty recipient channel, was incubated for 48 h. Subsequently, U937 cells were injected into the recipient channel and, 4 h after the cell injection, the internalization of U937-XP-secreted EVs by recipient cells was visualized using bright-field and GFP emission filters (arrowheads point at imaged EVs inside the recipient cells. Magnification = 1000×).