# Decellularized Skeletal Muscles Support the Generation of In Vitro Neuromuscular Tissue Models
## 1 Introduction  
In recent years, it has become evident that decellularized tissues represent a valuable tool in the field of tissue engineering. Along with their application in regenerative medicine strategies, decellularized scaffolds provide a 3D native-like environment for three-dimensional (3D) in vitro modeling of tissues or organs [1, 2, 3].  
The decellularized tissues are obtained via chemical, enzymatic, or physical methods to yield an acellular scaffold which preserves the complex 3D architectural organization, biochemical features, and biomechanical properties of the native tissue, including the extracellular matrix (ECM) [4]. The applicability of decellularized tissues and organs in translational studies is enhanced by the low immunogenicity of such scaffolds, enabling the use of xenograft materials, and by their structural and molecular properties that allow myogenesis and promote reinnervation and vascularization within the host [4, 5, 6]. An important aspect, specifically for skeletal muscle tissue engineering, is the ability to produce a sizable graft. This, in addition to recapitulation of structural and topographical complexity of the skeletal muscle tissue, had been a challenge, especially considering the necessity of such grafts for conditions associated with volumetric muscle loss [1].  
Due to their porosity and functionalization capacity, decellularized tissues can also be used as vehicles for drug and small molecule delivery for therapeutic purposes [7; 8; 9]. Recent advances in three-dimensional bioprinting technology enabled better control of the precise organization of larger in vitro hydrogel-based constructs, including hydrogels derived from decellularized tissues [10]. However, compared to dSkM, hydrogels alone offer suboptimal bioactivity and limited control of the overall 3D architecture that cannot reach the native-tissue complexity [1]. Besides their use in regenerative medicine, dSkM have also been utilized to reinstate the native tissue environment in vitro through tissue modeling. It is known that decellularized tissues act as a reservoir of signaling and attachment molecules that guide cell behavior native to the tissue of the decellularized construct [11]. Moreover, dSkM tissue has unique trophic and topographic properties which enhance repopulation, alignment and differentiation of myogenic cells [1]. These advantages pave the way to various applications of dSkM in vitro, e.g., investigation of cell viability and infiltration upon long-term culture [12; 13], evaluation of tissue ECM and drug interaction mimicking intramuscular injections [14], or the use of dSkM-based hydrogel for in vitro and in vivo studies [15; 16]. Our recent work has demonstrated that the dSkM retains a multitude of structural and other proteins that can enhance the repopulation of seeded myogenic cells [17]. In addition, we have demonstrated that dSkM has neurotrophic properties guiding the directionality of axonal projections when cocultured with organotypic spinal cord culture (oSpC) [17]. It is well known that, during development, regeneration, and homeostasis, skeletal muscle is highly dependent on the presence of neuronal input [18; 19]. Moreover, innervated 3D skeletal muscle constructs are crucial for disease modeling, drug screening, and molecular biology studies of neuromuscular healthy tissue and disorders [20; 21]. Based on our previous observations indicating the intrinsic ability of dSkM to attract neural axons [17], in the present study we wanted to investigate the possibility to develop an in vitro model of neuromuscular tissue using dSkM. Until now, to the best of our knowledge, there has been no previous report of neural and muscular co-culture within 3D dSkM scaffolds.