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. 2024 Sep 26;5(10):101748. doi: 10.1016/j.xcrm.2024.101748

Figure 3.

Figure 3

Assessment of morphology and barrier integrity of CD PDOs

(A and B) Representative images (A) of hematoxylin and eosin-stained FFPE-CD PDOs are shown from each clinical subtype of CD alongside healthy controls. Scale bar, 100 μm. L, lumen. Arrowhead, nuclear fragmentation (likely apoptotic bodies). Stacked bar plots (B) show the quantification of the proportion of each type of organoid structure in various CD subtypes (See Figure S7B for all CD subtypes combined; B [right], separated into CD subtypes). Statistical significance was assessed by one-way ANOVA. Only significant p values are displayed (n = 3–8 in each group). See Figure S7 for morphologic assessment by quantitative morphometrics using Imaris and lumen position/presence by light microscopy.

(C) Polarized monolayers of CD PDOs on transwells (enteroid-derived monolayers; EDMs) were fixed and stained for ZO1 (red), occludin (green), and DAPI (nuclei, blue) and analyzed by confocal microscopy. Representative fields are shown; individual red and green channels are displayed in grayscale. Asterisk, areas of impaired barrier. Scale bar, 50 μm.

(D) Electron micrographs of healthy and CD PDOs display apical cell-cell junctions. Red arrowheads, desmosomes. Scale bar, 2 μm.

(E) Electron micrographs of tight junctions in healthy and CD PDOs of various subtypes are shown. The boxed region on the left is magnified on the right. AV, apical villi; tj, tight junction; aj, adherens junction; d, desmosomes. Scale bars, 5 μm (top two panels) and 2 μm (bottom two panels).

(F–H) Plots display the quantification of no. of desmosomes/cell-cell contact (F), the length of TJ (G), and the frequency of abnormal defects/TJ structure (H) observed by TEM. Statistical significance was assessed by Welch’s t test (F, G) and Fisher’s exact test (H) (n = 7–13 fields analyzed in each subtype of PDO). See Figures S8A–S8C for the same analysis displayed as clinical subtypes of CD.

(I–L) Violin plots show the fold change in TEER across (I and K) and FITC-dextran leakage through (J and L) CD-EDMs compared to healthy EDMs. Data are displayed either as molecular (I and J) or clinical (K and L) subtypes. Statistical significance was assessed by Welch’s t test (I–K) and Mann-Whitney (L). Only significant p values are displayed (n = 5–9 subjects in each group, 2–5 repeats in each PDO). See Figures S8D–S8F for all CD subtypes combined. See Table S2 for subjects analyzed in each assay.