# Decellularized Skeletal Muscles Support the Generation of In Vitro Neuromuscular Tissue Models
## 3 Results
### Optimization and Characterization of 3D Neuromuscular Cultures  
It is reported that mechanical stretching promotes cellular myogenic differentiation and myotube alignment, maturation and function of newly formed myofibers [18; 19]. Based on that, and on our previous results [17], we developed a custom-made culture chamber in order to apply a linear constant stretching to the neuromuscular 3D construct (Figure 3A). Moreover, the chamber was designed to provide free access for imaging analysis. Therefore, polydimethylsiloxane (PDMS) was used to create the walls of the chamber located on the top of a glass coverslip (Figure 3B). Two PDMS pillars were also included within the chamber and used to tie the repopulated scaffolds via surgical stiches and to permit the constant stretching of the 3D culture system (Figure 3B). Since elongated stripes of dSKM were used as the scaffold in this experimental setup, we first monitored the ability of the oSpC to be firmly integrated into this new culture set-up. Then, human myogenic cells and muscular fibroblasts were seeded within the dSkM. The construct was stretched within the culture chamber and the oSpC was positioned onto the repopulated dSkM. Imaging analysis performed during the culture showed the progressive adhesion of the oSpC to the repopulated dSkM (Figure 3C). We then confirmed that our experimental setup allowed live 2-photon and second harmonic generation imaging analysis of living cells located deep within the dSkM (Figure 3D). Immunofluorescence analysis also indicated that myogenic (Desmin-positive) and neural (Tuj1-positive) cells were preserved in the developed culture set-up (Figure 3E). Importantly, contraction of the neuromuscular 3D model was observed in some areas of the construct and monitored by using 2-photon imaging analysis of  
Figure 1: Characterization of human myogenic cells and seeding into dSkM. (**A**) Representative images of immunofluorescence analysis performed for MyoG (green), Laminin (red), Nuclei (blue) in proliferating media (upper panel) or differentiating media (lower panel). Scale bars, 50 μm upper panels, 20 μm lower panels. (**B**) Representative bright field and calciea stereomicroscope (left panels), and 2-photon microscope (right panel) images of dSKM seeded with human myogenic cells and murine fibroblasts 21 days after seeding. Scale bars, 1 mm left panels, 200 μm right panel.  
calcein incorporating cells (Supplementary Video S2). To confirm the presence of MNs within our neuromuscular model, the 3D constructs were treated with Fluo-4 calcium dye, subjected to live imaging analysis, and stimulated with glutamate, the neurotransmitter of MN required for their depolarization, release of acetylcholine and induction of myofiber contraction [24]. Live imaging analysis performed with a 2-photon microscope of the 3D neuromuscular models located within the chamber revealed increased calcium flux after glutamate stimulation of cells located within the dSkM (Figure 3F).  
Figure 2: Decellularized scaffolds support 3D muscular and neural co-culture. (**A**) Schematic illustration of the experimental set-up. (**B**) Bright field (above) and calcein (below) stereomicroscope images of the dSkM repopulated with muscular and neuronal cells. Scale bar, 1 mm. (**C**) 2-photon microscope live imaging of calcein incorporation of the 3D neuromuscular construct. Scale bar, 100 \(\upmu\)m. (**D**) Representative images of immunofluorescence analysis performed for \(\beta\)III-Tubulin (Tuj1, green), Desmin (red) and F-actin (magenta). Nuclei were stained with Hoechst (blue). Scale bars, 250 \(\upmu\)m left panel, 50 \(\upmu\)m middle panel and 20 \(\upmu\)m right panel. (**E**) Left panel, representative Fluo-4 live imaging at two different time points. The arrows point at two myotubes (blue and red) that showed calcium waves, as quantified by the calcium fluorescent ratio (F/F0; blue and red, respectively). Scale bar, 300 \(\upmu\)m.