On-chip angiogenesis assay and vascular intraluminal pressure loading systems. a Microfluidic device mounted on a 35 mm glass bottom culture dish (left). Schematic illustration of top view of the device design (right). Five parallel channels (each 700 μm in width, a) were partitioned by microposts (100 μm window, b). b Schematic illustration of on-chip angiogenesis assay. In the on-chip angiogenesis assay, HUVECs seeded on Channel 4 migrate into the fibrin-collagen matrices filling Channel 3 and form angiogenic branches in response to soluble angiogenic factors secreted from human lung fibroblasts (hLF) in Channel 3. c Representative DIC images showing on-chip angiogenesis. Angiogenic branches which formed in Channel 3 before (Day 0) and 2 (Day 2) and 4 (Day 4) days after induction of angiogenesis are shown. d-f Confocal z-projection images and the orthogonal views (right in e) of angiogenic sprouts in an on-chip angiogenesis assay. d, the merged image of VE-cadherin (VE-cad, green) and DAPI (blue); e, the merged image of CD31 (white) and DAPI (blue) (left) and that of VE-cadherin (VE-cad, green) and DAPI (blue) (right); f, the merged image of VE-cadherin (VE-cad, green), actin (magenta), and DAPI (blue). Note that an angiogenic sprout has a lumen (e). An arrow in f indicates a tip EC. g Fluorescent images showing visualization of the vascular lumen by introducing FITC-dextran-containing angiogenic medium into channel 4. FITC-dextran diffused from the root of the angiogenic branch (left) and then toward the tip (right) for 1 sec through the lumen. h-l Devices for applying hydrostatic pressure to the lumen of elongating vessels. h, i Fixed type. Hydrostatic pressure (blue arrow in h) is applied to the lumens of angiogenic sprouts by placing capillaries filled with media (25 mm) in channel 4. Considering the negative pressure (red arrows in h and i, approximately 0.6 mmHg) generated by the capillary phenomenon (white line in i, water surface in culture dish), approximately 1.2 mmHg hydrostatic pressure is loaded on the lumens of angiogenic branches. j Variable type. Hydrostatic pressure was induced by height differences between the water surfaces in syringe 1 and syringe 2. White lines, water surfaces in syringes. k Schematic of hydrostatic pressure (arrows) loading system on microfluidic device (left, top view and right, side view). l Validation of the variable type of hydrostatic pressure loading system, using a differential pressure gauge. Data are presented as means ± s.e.m. (n = 5 devices examined over 5 independent experiments). Source data are provided as a Source Data file. Scale bars: 10 mm (left in a) and 200 μm (right in a), 100 μm (c, d), 20 μm (e, f). Reproduced from Choudhury, M.I., Li, Y., Mistriotis, P. et al. Kidney epithelial cells are active mechano-biological fluid pumps. Nat Commun 13, 2317 (2022). under Creative Commons Attribution 4.0 International License.