Figure 4. PGE₂-induced organoid swelling is dependent on Cl⁻.

The PGE₂-induced swelling of jejunal organoids was tested under normal, Cl⁻ reduced and Cl⁻ depleted buffer conditions. (a) Phase contrast view of the PGE₂-induced swelling in Ringer’s solution supplemented with standard Cl⁻ level (126.8 mEq). PGE₂ (10⁻⁹ M) was added to jejunal organoids for 30 min at 7 days after passage. Note that the swelling response is completely maintained. Scale bar represents 100 μm. (b) Phase contrast view of the PGE₂-induced swelling in Cl⁻-free Ringer’s solution. PGE₂ (10⁻⁹ M) was added to jejunal organoids for 30 min at 7 days after passage. Note that the swelling response is completely abolished by Cl⁻ depletion, but can be restored by the following addition of buffer supplemented with Cl⁻ (126.8 mEq). Scale bar represents 100 μm. (c,d) Time course experiment lasting up to 90 min showing the swelling response to PGE₂ under normal or Cl⁻-free Ringer’s solution. Jejunal organoids established from the uninflamed region of a CD patient (c) and those established from the healthy mucosa of a non-IBD patient (d) were subjected to the PGE₂-induced swelling (10⁻⁹ M) under normal or Cl⁻-free Ringer’s solution, and the response was quantified by the 3D-scanning system. (e,f) Quantification of the PGE₂-induced swelling under different Cl⁻ concentrations. Jejunal organoids established from the uninflamed region of a CD patient (e) and those established from the healthy mucosa of a non-IBD patient (f) were subjected to the PGE₂-induced swelling (10⁻⁹ M) under different Cl⁻ concentrations, and the response was quantified by the 3D-scanning system. Data are shown as the mean ± SEM of three independent wells. *indicates P < 0.05, ***indicates P < 0.0005, ****indicates P < 0.0001 as determined by two-sided Student’s t-test compared to the data of control (Adv DMEM; Advanced DMEM) or Cl⁻ 100% Ringer. ns indicates not significant. All results are representative of at least three independent experiments.