Fig. 2: Generation of anastomosed endothelial networks through functional vascularization of mesenchymal spheroids.

a Mesenchymal spheroids cultured on-chip under static or flow conditions. Representative images are shown at day 7 after seeding. b Angiogenesis Analyzer output of the morphology of the endothelial networks after a week of culture on-chip (n = 10 (static) and 8 (flow)). c Confocal z-stack maximum intensity projection of the three-dimensional endothelial network. d Gel embedded RFP-HUVEC cells in the main channel are shown in red, GFP-HUVEC cells from the mesenchymal spheroids are shown in green. Note the formation of structured endothelial networks over time that appear to be stable until the end of the observation period (day 13 after seeding). In a close-up view, anastomosis between RFP-HUVEC and GFP-HUVEC cells are indicated by white arrows (inset). e Schematic drawing depicting the cell culture configuration, with anticipated open connections at the fibrin gel interface allowing for microbeads perfusion (created with BioRender.com). f Maximum of intensity projection over a 71 images stack, highlighting the tracks of the microbeads passing through the interconnected network. See Supplementary Movie 1 and Supplementary Fig. 5 for raw movies and details. g Sum of the binarized and frame-color coded images from (f) showing time-resolved beads perfusion. See Supplementary Movie 2 for details. h Projections of maximum intensity over an image stack showing tracking of one individual microbead (red) passing through the endothelial network. The inset shows an assembled projection of three movies taken at the indicated area at higher magnification. For better visualization, the RFP-HUVEC cells are not shown in these images. See Supplementary Movie 3 for raw data. i Summary of different flow parameters measured in our organ-on-chip device as compared to in vivo physiological flow rates in human capillaries. Data represent mean values ± s.d. Beads were 1 µm (f), 4.8 µm and 0.5 µm (h and inset) in diameter. Scale bars, 400 µm (d), 200 µm (f and g), 100 µm (a, c and h), 50 µm (d (inset)) and 20 µm (h (inset)). Data represents mean ± s.d. Statistical significance was attributed to values of P < 0.05 as determined by unpaired t test (two-tailed). ***P < 0.001. Source data are provided as a Source Data file.