(A) Top (top panel) and side (bottom panel) view of the microfluidic wounding device. The wounding device is constructed of 2 layers, a top air layer (black) and bottom cell chamber (orange). The air layer has an air hole at one end where air pressure is increased by flowing air through a small tube connected to the hole. The ceiling of the cell chamber has a PDMS pillar that is lowered down on to the cells in the cell chamber when air pressure is increased in the air layer. (B) Cell chamber heights were measured by loading fluorescent beads into the device and allowing them to settle on the surfaces within the device (bottom, pillar, ceiling). Height was measured by scanning the cell chamber through the Z-axis and seeing where the beads had settled based on where they were in focus (bead sharpness). Cell chamber height within the device as measured by bead sharpness revealed a chamber of approximately 60 μm in height and a distance of approximately 15 μm between the bottom of the pillar and the bottom of the cell chamber. These distances provide ample room for cell growth while still limiting the z-space in which extracellular signals can diffuse. (C) The change in height of the ceiling of the cell chamber (orange) and the pillar (black) with increasing air pressure as measured by bead sharpness shows controlled movement of the pillar with increasing air pressure. Although the ceiling of the cell chamber also moves with the pillar, this does not interfere with cellular wounding. (D) MCF-10A cells were loaded in to the wounding device cell chamber and allowed to adhere in the presence of propidium iodide (PI) to measure cell viability within the device over time in static media conditions. Cells were able to adhere within 5 hr after which the media began to dry up and cells began to die indicating that cellular response following wounding can be measured for up to 5 hr in a static setting before cell death. (E) Wounding in non-static (with flow) and static (no flow) media conditions. Static media conditions were accomplished by placing a piece of tape over one of the ports of the wounding device during imaging. When flow was present, the signal went in the direction of the flow. However, when flow was not present, the signal propagated isotropically from the wound indicating that the response was a measurement of signal transfer and not simply due to the flow in the environment. (F) Ca2+ response according to distance away from the center of a 300 μm diameter wound shows reproducible wounding between 3 experiments as represented by 3 different colors. Cellular response is measured as the percentage of responding cells according to distance away from the wound. (G) Due to the layer-by-layer photolithography manufacture of the molds used to make the wounding device, the size of the wound can be controlled and reproducible. We were able to measure the single-cell spatiotemporal Ca2+ response in cells wounded by a 450 μm, 300 μm, and 150 μm wound. The single-cell responses for 4 different cells at varying distances from the wound are highlighted for the 450 μm wound with colorbar indicating fold maximum increase.