Fig. 2.

Biofabrication timeline. (A) Day 0: PDMS scaffold, spacers, and muscle-seeding molds are assembled on a glass coverslip. (B) Muscle strip bridging the 2 legs is formed during the first 2 d in culture then kept in differentiation medium for the following 6 d. (C) Optogenetic mouse ESCs are differentiated separately to obtain neurospheres, clusters of neural cells with photosensitive ChR2 ion channels and motor neurons expressing Hb9-GFP. (D) Day 8: Coculture is initiated by embedding neurosphere and muscle in a continuous ECM gel. (E) Day 11: Neurite outgrowth pattern after 3 d in coculture. Fluorescent intensity distribution around the neurosphere illustrates preferential neurite extension toward muscle strip. (F) Day 13 and onward: Optical stimulation of neurons induces muscle contractions. Evoked contractions are not observed in the presence of 25 µM curare. (Scale bars in B, C, and E: 250 µm.)