FIG. 6.

Calcium flux responses to stimulation in mono- and cocultured 3D human cell-derived engineered constructs. (A) Three-dimensional monoculture myoblast-seeded gels grown for 28 days in suspended 3D culture, incubated for 1 h with Fluo-4 (Fluo-4 Calcium Imaging Kit; Thermo-Fisher) at 37°C. Individual wells on 12-well plates, containing constructs containing 1 mL of Hank's Buffered Salt Solution were given a 100 mM acetylcholine chloride bolus and recording was started 5 s following the bolus. (B, D) Standard deviation Z-projection of AVI file with five frames per second and total recording length of 120 s. Image is shown in “Jet” lookup table (LUT) (ImageJ) with red shift indicating movement, and blue shift showing low deviation (left). Graph of three cells outlined as ROIs (yellow outline, left) shown in traces on right (C, E), with time 0 on chart indicating t = 5 s after acetylcholine bolus. Three traces are shown and are normalized to dark background (ROI # 4) as well as to baseline, taken as an average of 5 s at the end of recording. (F–I) Same as above with imaging of hiNSCs and hSKMs differentiated for 14 days before Fluo-4 incubation after l-glutamic acid stimulation. (J–L) hiNSCs incubated with CellTrace shown in coculture with glutamic acid stimulation before and after blockage with tubocurarine. (M) Relative fluorescence transient with identified neurons and myocytes before and after glutamate blocking agent. Scale bars 200 μm. ROI, regions of interest. Color images available online at www.liebertpub.com/tec