Figure 5:

Live-cell imaging of the response to RA in RFP-expressing fibroblasts that report RA signaling via GFP expression. The dual perfusion channel, cell-laden hydrogel was imaged to visualize constitutively active RFP and the reporter GFP expression after exposing the device sequentially to the following conditions: (A) perfusion of the left channel with 2 μM RA for 1 hour, (B) perfusion of the right channel with 2 μM RA for 1 hour, (C) perfusion of both channels with 2 μM RA for 1 hour, and (D) perfusion with standard media for 24 hours. Standard media was perfused for 24 hours between each RA condition. The GFP signal was localized to the region of the gel near the channels(s) perfused with RA, and the signal was reversible. (E) To confirm the cells remain viable in the entire region between the perfusion channels, RFP-expressing fibroblasts embedded in a hydrogel perfused with standard media for 14 days were stained with Calcein and Sytox blue (97% live cells).