Satellite cells delivered in their niche efficiently generate functional myotubes in three-dimensional cell culture

Biophysical/biochemical cues from the environment contribute to regulation of the regenerative capacity of resident skeletal muscle stem cells called satellites cells. This can be observed in vitro, where muscle cell behaviour is influenced by the particular culture substrates and whether culture is performed in a 2D or 3D environment, with changes including morphology, nuclear shape and cytoskeletal organization. To create a 3D skeletal muscle model we compared collagen I, Fibrin or PEG-Fibrinogen with different sources of murine and human myogenic cells. To generate tension in the 3D scaffold, biomaterials were polymerised between two flexible silicone posts to mimic tendons. This 3D culture system has multiple advantages including being simple, fast to set up and inexpensive, so providing an accessible tool to investigate myogenesis in a 3D environment. Immortalised human and murine myoblast lines, and primary murine satellite cells showed varying degrees of myogenic differentiation when cultured in these biomaterials, with C2 myoblasts in particular forming large multinucleated myotubes in collagen I or Fibrin. However, murine satellite cells retained in their niche on a muscle fibre and embedded in 3D collagen I or Fibrin gels generated aligned, multinucleated and contractile myotubes.