FIG. 6.

Long term cell culture and myoblast differentiation into myotubes. (a) Day 0: One hour after cell seeding, C2C12 myoblasts were adherent and spread inside the Matrigel-coated PDMS trench. Few cells attached on the outside of the trench, showing the effectiveness of the Pluronic F127 cell-repellant coating. Cells grew confluent and filled the trench by Day 3, during which the growth media were changed to differentiation media containing low serum (not shown). (b) At Day 6, 3 days after change to differentiation media, myoblasts fused and formed elongated myotubes. (c) Day 14: Mature myotubes are shown within a 100 μm wide trench. Note that cells outside of the trench have died and detached. (d) On-chip differentiation of C2C12-derived myotubes at Day 9 showed their elongated, mature phenotype. (e) Elongated mature myotubes at Day 8 within a 100 μm wide trench, and (f) expression of mature acetylcholine receptor “pretzel” patterns labeled by fluorescent bungarotoxin.