File Name : figure_s1.tif

Caption : supplementary figure 1. characterization of su-8 masters and µgroove substrates. (a) experimental dimensions of 5:1, 5:2, 7:1 and 7:2 µgroove geometries. for si masters, five pictures were taken along the samples with the profilometer to consider possible inhomogeneities inherent to the fabrication process. values are expressed as mean ± sd. (b) representative images of su-8 masters characterized with an optical profilometer. (c) representative brightfield images of µgroove structures (5:2, left panel). in some cases, delamination artifacts were observed causing shape distortion or partially closed structures (right panel). scale bar: 100 µm.

File Name : figure_s2.tif

Caption : supplementary figure 2. human immortalized skeletal muscle media components.

File Name : figure_s3.tif

Caption : supplementary figure 3. immortalized human myotubes development (8220). (a) myoblast fuse and differentiate into isolated contractile myotubes within µgrooves of different geometries. differentiation was promoted 24 hours after seeding (0 days in differentiation, 0 dpd). (b) representative fluorescent images of dapi stained nuclei 4 hours post-seeding. scale bar: 100 µm.

File Name : figure_s4.tif

Caption : supplementary figure 4. experimental set-up. chronical electrical stimulation promotes myotubes differentiation and maturation, thus increasing their contractile properties. video recording of the myotubes reaching twitch and tetanic contractions was perform at 4-dpd and analyzed with musclemotion software. from these curves, variables like time to peak (ttp), peak amplitude (pa) and half relaxation time (rt50) were determined. 

File Name : figure_s5.tif

Caption : supplementary figure 5. myotubes excitability. (a) myotubes were electrical stimulated at increasing voltages (4-40 v). random fields have been selected in the case of the controls and full grooves in the case of the different geometries. we calculated the percentage of responding myotubes under electrical stimulation as the ratio of those contracting to those not contracting in the selected field. all myotubes grown in the ngc responded at 8 v while for the ones grown within µgrooves, this response was highly dependent of voltage, increasing this parameter increase the number of myotubes that contract. (b) µgrooves were simulated in ansys electronics software to study the electric field behavior in the µgrooves. (c) electric field simulation for 8, 10 and 20 v in µgrooves and ngc. voltages of 4 and 40 were not analyzed because, for the first case, we obtained no response in either µgrooves or ngc, and for the second case, all myotubes responded to this voltage. width and length are displayed to check both orientations. 

File Name : figure_s6.tif

Caption : supplementary figure 6. spontaneous contraction was observed after chronical stimulation with a frequency of approximately 1 hz. in some cases, these spontaneous contractions were observed while evaluating the myotubes under increasing frequencies (yellow arrows).

File Name : figure_s7.tif

Caption : supplementary figure 7.  coefficient of variation percentage (cv%) of μgrooved myotubes and non-grooved controls (ngc) for different assays. (a) myotube width (µm). (b) coefficient of variation (%cv) of myotube width datasets. the %cv decreased by 59.56%, 27%, 59.76% and 13.54% for 5:1, 5:2, 7:1 and 7:2 µgrooves, respectively, compared to ngc. dots  represent values from different µgroove geometries. (c) twitch and tetanic contractility measured by peak amplitude (a.u., arbitrary units; left panel), and twitch kinetics for time to peak (ttp) and half-relaxation time (rt50; right panel) (d) resting cytosolic ca2+ (nm). (e) cytosolic calcium fluxes in response to twitch and tetanic stimuli. statistical analyses performed are detailed below each table.