# Decellularized Skeletal Muscles Support the Generation of In Vitro Neuromuscular Tissue Models
## 4 Discussion and Conclusions  
The development of neuromuscular in vitro models implies a recapitulation of the native-like structural and functional complexity of the interactions existing between the two tissues. The structural architecture relies on 3D organization of the construct and its functionality depends on the degree of maturation of the cellular components within the construct. The advantages related to the use of dSkM in reconstructing skeletal muscle tissue [4; 5] and our own previous results from studying dSkM neurotrophic properties [17] drove our interest in the development of a 3D in vitro neuromuscular model based on a repopulated dSkM scaffold. Figure 3: Characterization of 3D neuromuscular construct cultured in custom-made PDMS chamber. (**A**) Schematic illustration of the experimental set-up within the custom culture chamber. (**B**) Representative image of muscle scaffold allocated into the PDMS chamber. Scale bar, 1 mm. (**C**) Bright field high magnification images of organotypic spinal cord section 1, 2 and 5 days after seeding. Scale bar, 500 μm. (**D**) Representative 2-photon and second harmonic generation imaging of Fluo-4-positive (green) cells within dSkM ECM (white). Scale bar, 500 μm. (**E**) Representative images of immunofluorescence analysis for \(\beta\)III-Tubulin (Tuj1, green) and nuclei (blue) of the 3D neuromuscular constructs. Scale bars, 500 μm, left and right panels; 200 μm, middle panel. (**F**) Left panel, Fluo4 two photon-mediated calcium imaging of 3D constructs before (left) and after (right) glutamate stimulation. Right panel, Scale bar, 500 μm. Right panel, quantification of calcium fluorescent ratio (F/F0) of images acquired before and after glutamate stimulation. Data are shown as mean \(\pm\) s.d. of three independent replicates; \({}^{**}\), \(p<0.05\). Our initial evaluation of repopulated dSkM confirmed the ability of the scaffold to support the migration and differentiation of myogenic cells. In addition, the scaffold enabled cell culture for up to 21 days and supported the co-culture of both the myogenic and neural compartments. Based on the evaluation of the physiological activity of our in vitro model and its imaging analysis, dSkM not only allowed the assisted juxtaposition of different cell types, but also nurtured the maturation of myogenic cells, resulting in the generation of calcium spikes. Our optimized, custom-made chamber introduced yet another advantage to the development of our model. Specifically, the suspension of the construct on PDMS pillars allows for easer live imaging analysis of the 3D neuromuscular construct. Stretch has important implications for alignment, differentiation, and maturation of the myogenic cell populations in vivo and in vitro [18; 19]. Indeed, during co-culture in chambers, we observed spontaneous contractions within the scaffold, indicating enhanced maturation of myogenic cells and possible communication with the neural counterpart. In the future, further analysis will be required to evaluate the presence of structurally and functionally competent neuromuscular junction (NMJ) and their state of maturation. Further design iterations will be focusing on integrating an adjustable tensioner, allowing to adjust the level of stretching applied to the constructs. Overall, our findings provide evidence of the possibility to develop functional neuromuscular in vitro constructs using dSkM as a 3D scaffold. We also acknowledge that our study has certain limitations. The major limitation would be the investigation of interactions of cell types derived from different species. While the most optimal representation of neuromuscular tissue would be a combination of human-only cell sources, we have previously proven in other engineered models that a xenogeneic in vitro system can replicate biological models with fidelity [25]. Current developments in differentiation methods have enabled the generation of human-based motor neuron cell cultures which could be utilized to build humanized in vitro models in the future.