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. 2020 Dec 9;33(3):2005476. doi: 10.1002/adma.202005476

Figure 3.

Figure 3

Human‐derived rECM hydrogels as bioinks for airways. a) H&E staining of HBECs 28 days after seeding on top of alginate and rECM hydrogels. Scale bar: 10 µm. b) Immunofluorescence stainings of KRT5, p63, MUC5AC and acetylated‐α‐tubulin positive cells 28 days after seeding HBECs on top of alginate and rECM hydrogels (n = 3 patients per group) Scale bar: 20 µm. c) SEM images of ciliated cells present in HBECs seeded on top of alginate and rECM hydrogels on day 28 (n = 3 patients per group). Scale bars: 3 µm. d) Changes in gene expression of HBECs seeded on top of alginate and rECM hydrogels and lifted to ALI on day 7 showing the fold change compared to day 0 of differentiation genes on day 7, 14, 21, 28, and 35 (increase in red and decrease in blue) (n = 3 patients per group (P1, P2, and P3)). e) Live–dead imaging and quantification of HBECs 3D bioprinted in alginate and rECM hydrogels right after bioprinting. The large panel is the top‐down view after seeding with side view below (live cells in yellow and dead cells in purple) (n = 3 patients per group). Scale bars: 100 µm. f) Side view of disk constructs with live cell tracker imaging of proliferating 3D bioprinted HBECS in alginate and rECM hydrogels on day 0, 7, and 35 (n = 3 patients per group). Scale bar: 200 µm. g) Cell sedimentation confocal images and calculated sedimentation coefficient (δ) of human lung epithelial A549 cells encapsulated in DMEM‐F12 media, alginate, and human derived rECM solution for six hours (n = 3 per group). Scale bar: 500 µm. h) CAM assay images of human‐derived rECM hydrogels and parafilm as negative control on day 10 and change in blood vessel on day 10 compared to day 6 normalized to parafilm in human‐derived rECM hydrogels (n = 7–10 per group). Scale bar: 1 mm. i) 3D rendering of bioprinted airways. j) 3D bioprinted airway with human lung smooth muscle cells (HLSMCs) in yellow in the outer perimeter and human bronchial epithelial cells (HBECs) in blue in the lumen on day 0 and on day 28 (n = 3 patients per group). Dotted white lines indicate the inner lumen. Scale bars: 500 µm. k) 3D bioprinted airway with acetylated‐α‐tubulin staining in red on day 28 (n = 3 patients per group). Inner lumen appears closed due to a processing artefact. Scale bar: 500 µm (lower) and 25 µm (upper).