38 Chapter 2 Figure 1. Amino acid starvation of mature hepatic organoids leads to functional changes without profound morphological changes. (A) Representative bright-field images of control and starved mature hepatic organoids prior (T0H) and after 96 h (T96H) amino acid starvation. Scale bar, 1 mm. (B) Size of organoids in complete culture medium (control, grey) or amino-acid free medium (starved, blue), expressed as size of an organoid at every time point of interest. Data based on 3 sets of independent experiments in which 25 randomly selected organoids were tracked. Error bars indicate SEM. (C) Albumin released in supernatant by organoids that were kept in complete medium throughout (control, grey), starved from 0h to 48h and re-supplemented with complete medium from 48h to 96h (re-supplementation, orange), or kept in complete medium and starved from 48h to 96h (starved, blue). Change in experimental conditions over time are indicated by colored bar chart. Data represents 3 biological replicates from independent experiments. Error bars indicate SEM. (*P<0.05, generalized estimating equation with Tukey adjustment for post-hoc pairwise comparison). (D) Representative immunofluorescence images of control and starved mature hepatic organoids (40x magnification). BODIPY staining of fat droplets in green with Hoechst nuclei counter stain (blue). Scale bar, 50 μm. (E) Triglyceride levels in control, starved and re-supplemented organoids (as per colour legend). Data represents 5 biological replicates from independent experiments ± SEM (*P<0.05, ordinary one-way ANOVA with Tukey’s post hoc test). Impact of amino acid starvation on structure and function of small intestinal organoids After 48 hours of amino acid starvation, intestinal organoids were significantly smaller than non-starved organoids (Figure 2a, b and Supplementary Figure 4a). In addition, amino-acid-starved organoids displayed crypt atrophy and had substantially fewer crypts than control organoids (Figure
RkJQdWJsaXNoZXIy MTk4NDMw