Decellularized skeletal muscles display neurotrophic effects in three-dimensional organotypic cultures

Abstract

Skeletal muscle decellularization allows the generation of natural scaffolds that retain the extracellular matrix (ECM) mechanical integrity, biological activity, and three-dimensional (3D) architecture of the native tissue. Recent reports showed that in vivo implantation of decellularized muscles supports muscle regeneration in volumetric muscle loss models,

Decellularized Skeletal Muscles Support the Generation of In Vitro Neuromuscular Tissue Models

“…A mixture of 7 × 10 5 human skeletal myoblasts and 3 × 10 5 mFBs was injected into the scaffold section, put on a glass coverslip using insulin syringes, and kept in culture for 3 weeks in proliferating medium. Organotypic spinal cord 3D culture was prepared as previously described [17]. For co-culture experiments, isolated oSpCs were dissected into three segments of ~1 × 1 × 2 mm format of apical, central, or caudal identity.…”

“…Based on our previous results and on the neurotrophic properties of dSkM [16,17], we generated the neuromuscular construct by co-seeding human myogenic cells and murine fibroblasts into the dSkM together with oSpC onto the scaffold (Figure 2A). Calcein liveimaging performed at the fluorescence stereomicroscope, confirmed the presence of viable cells after 21 days of co-culture (Figure 2B).…”

“…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].…”

“…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.…”