Fig 1.
Overview of the cell seeding strategy for HIE-derived 3D intestinal constructs.
(a, b) HIEs isolated from human patients are cultured in the Matrigel. (c) HIEs were enzymatically digested to obtain Singlet/doublet cells. (d) HIE-derived cells were seeded onto the luminal surface of a 3D tubular silk scaffold, while H-InMyoFibs were delivered into the spongy silk scaffold bulk. The constructs were cultured in differentiation medium for at least 3 days to induce intestinal epithelial differentiation. (e) SEM showed the microvilli brush border formation at the apical cell surface. Scale bar, 10μm. (f) Highly organized ZO-1 chicken wire pattern staining in differentiated HIE-derived epithelium on 3D scaffolds. Scale bar, 15μm. (g) ALP staining on the epithelial cells were observed on the epithelium. Scale bar, 250μm.
Fig 2.
The differentiation of 3D intestinal epithelia.
(a, b) General seeding cell seeding strategy for hInEpiC-derived and cell line-derived 3D intestinal constructs. (c-n) Immunohistological stainings of SI (sucrose-isomaltase, c, g, k), MUC-2 (Mucin 2, d, h, l), Lysozyme (e, i, m) and ChgA (Chromogranin A, f, g, n) showed the location of enterocytes, Goblet cells, Paneth cells, and enteroendocrine cells in differentiated HIE-derived, hInEpiC-derived cell line-derived epithelia. Scale bar, 25μm. (o) Relative mRNA expression levels of different markers of differentiated intestinal epithelia derived from the three different cell sources. The fold-change in mRNA expression was compared with cell line-derived 3D constructs at day 1 post cell seeding.
Fig 3.
Gene expression levels of four intestinal epithelial cell markers, including SI (a), Muc2 (b), Lysozyme (c) and ChgA (d), functional epithelium markers, including ZO-1 (e), Villi (f), and ALP (g), and an intestinal stem cell marker, Lgr5 (h) were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) overtime in cultures. Data is presented as mean±SEM, n = 5 in each group, p<0.001. The fold-change in mRNA expression was compared with cell line-derived 3D constructs at day 1 post cell seeding.
Fig 4.
The oxygen concentration profiles of HIE-derived (a), hInEpiC-derive (b), and cell line-derived (c) tissues 3D tissues were measured using an oxygen meter.
Fig 5.
Human antibacterial response RT2 Profiler™ PCR arrays.
(a-c) Heat-map comparison of 84 antibacterial genes in HIE-derived (a), hInEpiC-derived (b), and cell line-derived (c) epithelia after exposure to E. coli. for 4 hours. Genes were displayed for fold-change variation in respect to their uninfected control groups and colored by their normalized expression value (red: high expression; green: low expression). (d-f) Scatter plot charts. Genes upregulated with fold change greater than 4 and are showed as red dots; genes with fold change less than 4 are showed as green dots; unmodulated genes are showed as black. (g) Heatmap detail displayed all upregulated genes for HIE-derived, hInEpiC-derived, and cell line-derived epithelia after E. coli infection.