Figure 9. Smooth muscle cells mediate Ca²⁺ synchronization between organoids.

(A) Diagram of a three-well hydrogel in which one organoid was placed per well. The three wells were connected with narrow channels, and this gel mold does not allow organoidal bodies to fuse to each other, but allows them to extend/migrate protrusions/cells through the channel (B). (C) After 3 days, the three organoids displayed synchronization of Ca²⁺ dynamics. (D) Some organoid-derived cells crawled out from the wells and covered the top surface of the hydrogel, resulting in bridging the three unfused organoids. (E) Diagram of a three-well hydrogel without channels. (F) The surface-covering cells were identified as SMCs (αSMA-positive, c-Kit-negative). (G) In the hydrogel without channels but with surface-covered SMCs, Ca²⁺ dynamics in the three organoids were synchronized. (H) The Ca²⁺ synchronization shown in (G) was not altered by treatment with 18β-GA. Blue planes indicate focal planes. Scale bars: 50  µm (B); 100  µm (C, D, F, G, H).

Figure 9—video 1. Ca²⁺ transients in three gut contractile organoids in three-well hydrogel with channels.

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Time-lapse images were obtained with 700 ms intervals for 5 min. This video corresponds to Figure 9C. Scale bar: 100 µm.

Figure 9—video 2. Ca²⁺ transients in three gut contractile organoids in three-well hydrogel without channels.

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Time-lapse images were obtained with 450 ms intervals for 5 min. This video corresponds to Figure 9G. Scale bar: 100 µm.

Figure 9—video 3. Similar assay to Figure 9—video 2 with 18β-GA administration.

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Time-lapse images were obtained with 700 ms intervals for 5 min. This video corresponds to Figure 9H. Scale bar: 100 µm.