40 Chapter 2 Figure 2. Amino acid deprivation induces reversible crypt atrophy, compromised growth and barrier dysfunction in intestinal organoids. (A) Size over time of organoids grown in complete culture medium (control, grey), amino-acid-free medium (starved, blue) or amino-acid-free medium for 48 h followed by complete culture medium for 48 h (re-supplementation, orange). Data represents the growth of 25 measured organoids in each of the 3 biological replicates from independent experiments. Error bars indicate SEM (* indicates comparison between control and starvation, # indicates comparison between starvation and re-supplementation) (*** P< 0.001, **P< 0.01, *P<0.05, ### P< 0.001, ##P< 0.01, # P<0.05, generalised estimating equation with Tukey adjustment for post-hoc pairwise comparison). (B) Representative brightfield images of control and starved intestinal organoids at the start and after 48 h. Scale bar, 200 μm. (C) Representative bright-field images show an individual small intestinal organoid grown in complete culture medium for 3 days (C1), switched to amino acid free medium for 48 h (C2 and C3), and then placed in complete culture medium for 48 h (C4 and C5). Arrowheads indicate individual crypt domains. (D) Average number of crypts per organoid over time for control, starved and re-supplementation. Data represents the average of 25 measured organoids in each of the 3 biological replicates from three independent experiments at 5 time points. Error bars indicate SEM (** P < 0.01, *** P <0.001, generalised estimating equation with a Poisson variance to mean relation and Tukey adjustment for post-hoc pairwise comparison.
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