Journal: iScience

Article Title: Proteomic and functional profiling of suspended jejunal enteroids reveals pharmacokinetic fidelity to native intestine

doi: 10.1016/j.isci.2026.117654

Figure Lengend Snippet: Epithelial barrier integrity assessment in 3D enteroids in suspension (A) Expression of important tight junction proteins in MBO, AO, and BO as compared to primary enterocyte and jejunal mucosa in vivo levels. Data are shown as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (DEqMS); ND = not detected; NQ = not quantified. Data from different donors is shown ( n = 4 enteroid donors Hu18-9jej, Hu22-2jej, Hu22-3jej, Hu22-5jej; n = 4 enterocyte donors Hu21-7jej, Hu21-14jej, Hu22-3jej, Hu22-5jej and n = 5 jejunal mucosa donors Hu21-7jej, Hu21-14jej, Hu22-2jej, Hu22-3jej, Hu22-5jej). (B and D) Representative time-lapse series showing suspended (B) AO and (D) BO enteroids that were incubated in medium containing lucifer yellow (LY, 100 μM) at 37°C to assess intact epithelial barrier over a time period of 90 min. AO enteroids were imaged live by confocal microscopy, and LY fluorescence intensity was determined within the outline of the enteroid and background. Scale bars, 50 μm. Images are representative of 44 AO and BO enteroid observations. (C and E) Quantification of LY fluorescence intensity within enteroids was normalized to background. For the experiment, an average of 44 enteroid observations per time point was made; the Kruskal-Wallis test showed no statistically significant difference between time points. Data are shown as median ± IQR. Data are representative as one of two independent experiments. (F and G) Incubation with Ca 2+ chelator EDTA disrupts paracellular barrier integrity in (F) AO and (G) BO enteroids. The LY permeability assay was used to evaluate apical-out enteroids treated with EDTA for 30 min. Enteroids were imaged as described in (B and D) using live-cell confocal microscopy. Scale bars, 50 μm. The corresponding quantification of LY fluorescence intensity within untreated enteroids (incubation with LY for 30 min) and enteroids treated with EDTA (additional incubation for 30 min with EDTA) was normalized to background. Data are shown as median ± IQR. An average of 44 enteroid observations were analyzed; ∗∗∗∗ p < 0.0001 (Mann-Whitney U test). (B–G) Enteroids from donor Hu18-9jej were used. See also .

Article Snippet: Ethylenediaminetetraacetic acid (EDTA) , MP Biomedicals , Cat #: 2820549.

Techniques: Suspension, Expressing, In Vivo, Incubation, Confocal, Microscopy, Fluorescence, Permeability, MANN-WHITNEY