Fig 2: Seeding of endothelial cells and podocytes in OrganoplatesTM and generation of the GOAC. a Representation of seeded hGEC in OrganoplateTM. b Confocal Z-stack image showing formation of capillary-like structure. PhaseguideTM components (yellow lines) can be easily identified within the panel. Channel E is filled with collagen I (visible thanks to autofluorescence in the red channel) while channel C confirms formation of a capillary-like structure by a continuous monolayer of hGEC (stained with CD31, Alexa-555, red). c, d confocal image for CD31 (Alexa-555, red, c) and for WGA (Rhodamine, red, d) in hGEC after 28 days of culture. e Representation of seeded podocytes in OrganoplateTM. f–h Confocal image for nephrin (NPHS1-FITC, green) in hAKPC-P (f), in hiPOD (g), and hpPOD (h) seeded on the channel C after 28 days of culture. Nephrin expression is present on the level of cell–cell contact (arrow). i Representation of seeded of podocytes and hGEC in OrganoplateTM. j–l Confocal image for nephrin (NPHS1-FITC, green) and CD31 (Alexa-555, magenta) in hAKPC-P + hGEC chip (j), in hiPOD + hGEC chip (k), and hpPOD + hGEC chip (l) after 28 days of culture. All three podocyte lines form a continuous layer, distinguishable from the hGEC layer. m Representation of a GBM-like structure in OrganoplateTM. n–v Confocal image for COL4A4 (Alexa-555, red), for COL4A12 (Alexa-555, red), and for LAMA5A (Alexa-555, red) in hAKPC-P + hGEC chip (n–p), in hiPOD + hGEC chip (q–s), and hpPOD + hGEC chip (t–v) after 28 days of culture. hAKPC-P + hGEC chip and hpPOD + hGEC chip show de novo generation of GBM, which is less evident in the hiPOD + hGEC. Nuclei are stained with DAPI (blue). All pictures: scale bar = 50 µm; except bottom panel in f–l with scale bar = 25 µm bar. w–y Western blot analysis for COL4A3 (25 kDA, monomeric form; 50 kDA, dimeric form), LAMA5 (70 kDa), and beta actin (40 kDa) in hAKPC-P + hGEC (w), hiPOD + hGEC (x), and hpPOD + hGEC (y) GOAC. Positive control: human whole-kidney lysate. Western blot images were cropped to show the relevant bands and improve clarity. Uncropped and unprocessed scans for western blotting analysis are provided in Supplementary Fig. 11

Fig 3: Characterization of cellular lines and description of the microfluidic chip. a–i Flow cytometry for WT1 (PE, red) in hAKPC-P (a), hiPOD (b), and hpPOD (c); for nephrin (NPHS1-FITC, green) in hAKPC-P (d), hiPOD (e), and hpPOD (f) and for CD31 (FITC, green) in hAKPC-P (g), hiPOD (h), and hpPOD (i). All three lines show almost 100% expression of podocyte markers and absence of endothelial marker confirming their podocyte phenotype. j–l Flow cytometry for WT1 (PE, red; j), for nephrin (NPHS1-FITC, green; k), and for CD31 (FITC, green; l) in hGEC show low expression (<1%) of podocyte markers and higher expression of endothelial marker confirming their endothelial phenotype. m–r Flow cytometry for WT1 (PE, red) in HuLEC (m) and hFIB (p), for nephrin (NPHS1-FITC, green) in HuLEC (n) and hFIB (q), and for CD31 (FITC, green) in HuLEC (o) and hFIB (r). HuLEC and hFIB are both negative for podocyte makers, HuLEC are positive for endothelial marker while hFIB are negative. Unstained control is shown in red while stained sample is shown in light blue. s, t The chip is a microfluidic layer sandwiched between two 175 µm glass plates (OrganoPlateTM platform, courtesy of MIMETASTM, panel s). The three-channel version of the OrganoplateTM comprises 40 networks on one 96-well format plate. t The central section is subdivided into three channels by a system known as PhaseGuide™, a thin 30 µm tall ridge that acts as a pinning barrier for incoming fluids23. Following the filling of channel E with collagen I, channel C (representing the vascular space) is seeded with cells and then filled with growth medium; the channel F (representing the urinary space) is where the filtrate is collected. Cross-section depicts patterning of cells and collagen within the GOAC. Flow of culture medium is achieved by leveling between the media reservoirs of the lanes C and F. The platform is placed on an interval rocker, with an angle to assure leveling. By changing the angle of the platform (rocker settings: interval = 8 min, angle = 7°), the direction of fluid flow is reversed