File Name : supp figure 1.tif Caption : supplemental figure 1: millifluidic flow device set-up. the design of the flow system allowed for recirculation of cell media to conserve media volume. image created in biorender. File Name : supp figure 2.tif Caption : supplemental figure 2: bbb model permeability preliminary studies for work reported herein, and previously published results. (a) comparison of the measured fluorescent intensity units for the bbb model in which hbmecs were plated in the apical compartment of the transwell insert and acs and pcs on the basolateral side of the transwell insert (epa) vs. the bbb model in which pcs and acs were plated in the apical compartment and ecs on the basolateral side (ape). the results indicated no apparent difference in permeability when the results were compared. (b) teer values of static vs. flow conditions, demonstrating high variability of the teer measurements. due to the large variability in results produced by the teer chopstick electrode system, dextran permeability assays were the preferred method for quantifying barrier integrity, as reported herein. (c) prior published bbb permeability levels for immortalized, primary, and inducible pluripotent stem cell derived (ipsc-derived) ecs in monoculture; bi-culture with acs or pcs; or in tri-culture with both acs and pcs. this plot is re-used from the work of destefano et. al. 95 which was published as “an open access article distributed under the terms of the creative commons cc by license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.” File Name : supp figure 3.tif Caption : supplemental figure 3: human brain microvascular endothelial cells align in the direction of flow preferentially at lower cell densities. fluorescent f-actin staining (top left) demonstrates preferential parallel cell alignment at low cell density but less alignment at high cell density. quantification (top right) is shown of hbmec cell alignment after 24 hours of flow exposure at 12 dynes/cm2 in a parallel plate flow chamber at either low or high relative cell densities. specific results appear in the first table (at the center of this supplemental figure 3), where data is presented as the percentage of cells aligned at the specified angle in mean ± sem. the second table (at the bottom of this supplemental figure 3) shows the statistical analysis results for hbmec cell alignment under flow, as observed using the parallel plate flow chamber. File Name : supp table 1.tif Caption : supplemental table 1: summary of shear stresses used in similar in vitro bbb models. citing numerous examples of the current body of work in which there is a wide range of shear stresses applied when modeling brain microvasculature in vitro. our future studies will investigate the wide variety of shear stresses to identify which flow rates elicit physiological and pathological cellular responses. File Name : supp table 2.tif Caption : supplemental table 2: expression of permeability regulating proteins in e, ep, ea, and epa samples as determined by western blotting. (a) normalized mean ± sem data corresponding to figures 6 a and b. (b) statistical analysis results for western blot protein expression in e, ep, ea, and epa samples corresponding to figures 6 a and b. File Name : supp table 3.tif Caption : supplemental table 3: expression of tight junction proteins in static versus flow conditions for either endothelial monolayers (e) or ec/pc/ac co-cultures (epa) as determined by western blotting. data corresponds to figures 6 c and d.