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. Author manuscript; available in PMC: 2016 Feb 26.
Published in final edited form as: Nat Biotechnol. 2015 Jul 13;33(8):845–852. doi: 10.1038/nbt.3275

Figure 3.

Figure 3

Functional characterization of CLC organoids (a) Representative images demonstrating the MDR1 fluorescent substrate Rhodamine123 detected in the lumen of CLC organoids, confirming MDR1 functionality. Scale bars, 50μm. (b) Fluorescence intensity measurements along the area indicated by the red line. (c) Mean intra-luminal fluorescence intensity normalized over background, in the presence (+VER) or absence (−VER) of verapamil, n=599 measurements, P=2.99×10−5 (2-tailed t-test). (d) Representative images demonstrating active export of the fluorescent bile acid cholyl-lysyl-fluorescein (CLF) from the lumen of CLC organoids compared to controls loaded with Fluorescein Isothiocyanate (FITC). (e) Fluorescence intensity along the area indicated by the red line. (f) Mean intraluminal fluorescence intensity normalized over background, n=1163 measurements, P<1×10−18 (2-tailed t-test). The data shown in panels a-f is representative of 3 different experiments. (g) Fluorescence intensity measurements (top) and representative fluorescence microscopy images (bottom) of CLC organoids loaded with the calcium indicator Fluo-4, demonstrating an increase in intracellular calcium levels following stimulation with ATP and acetylcholine. Plated primary cholangiocytes stimulated with ATP are used as a positive control. Grey area represents 1 SD, n=3. (h, i) Fold change over starting number of cells (h) and IF analyses for the proliferation marker Ki-67 (i) in the presence and absence of VEGF for 5 days, demonstrating that VEGF promotes CLC proliferation. Prim. Chol.: Plated primary cholangiocytes, n=10, p=4.77×10−17 (CLCs), p=4.63×10−17 (Prim. Chol.) (2-tailed t-test). (j) CLC organoids exhibit GGT activity. MEF: Mouse Embryonic Feeders, n=3, p<0.0001 for all comparisons (one-way ANOVA with Dunnett correction for multiple comparisons). (k) ALP staining revealing ALP activity in CLC organoids. Top: Photographs of stained wells (Scale bars:1cm). Bottom: Brightfield microscopy images, n=3. Scale bars:100μm. Data representative of multiple lines (Figure 6a-6b, Supplementary Figure 5). All error bars represent s.d. Asterisks (****) in panels c, f, h and j indicate statistical significance of the differences demonstrated (P < 0.0001).