Fig. 2
From: A glomerulus-on-a-chip to recapitulate the human glomerular filtration barrier
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
