Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC165
Development of physiologically correct neural inputs for an in vitro model of skeletal muscle.
A. Smith1,3, V. Mudera2, L. Greensmith1, M. Lewis3
1. Institute of Neurology, UCL, London, United Kingdom. 2. Institute of Orthopaedics and Musculoskeletal Science, UCL, London, United Kingdom. 3. Muscle Cellular and Molecular Physiology Research Group, University of Bedfordshire, Bedford, United Kingdom.
The ability to engineer physiologically correct in vitro models of skeletal muscle is likely to play an important role in the future investigation of skeletal muscle development, physiology and pathology. Although current models develop a physiological architecture and contractile properties similar to those of the nascent tissue, there is now a drive to promote maturation towards a mature phenotype, which necessitates integration of a neural input into the model. This work seeks to establish a neural input for an established skeletal muscle model through the integration of primary motoneurons with cultured myotubes. As well as promoting maturation towards an adult phenotype, reliable neuromuscular junction (NMJ) formation in 3D culture could have substantial benefits in the study of neuromuscular disease and the testing of novel therapeutic agents. Methods for establishing the muscle model are detailed elsewhere (Cheema et al., 2003) and rely on the inherent ability for cells seeded in a 3D matrix to self orientate. Within this model, contraction of the seeded cells against uniaxial fixed points leads to formation of isometric tension sufficient to promote the reorganisation of the cells along lines of strain. Constructs were cultured with primary embryonic rat motoneurons for 14 days before being prepared for analysis. Images from stained sections of the constructs demonstrate the improved functional architecture of the 3D muscle constructs compared to 2D and the similarity to that observed in in vivo muscle controls (n=9). Close association of developing neurites with underlying myotubes is clear and there is evidence of formation of structures in vitro that closely resemble NMJs in vivo. Gene expression analysis suggests a 2-3 fold increase in expression of markers of muscle maturation, such as myosin heavy chain isoforms, troponin and acetylcholine receptor subunits, in 3D co-cultures compared with muscle only controls (n=3). Current efforts are focused on improving the incidence of synapse formation and further improving myotube-motoneuron interaction in vitro. Once optimised, this development in muscle modelling should have significant repercussions in the future investigation of neuromuscular interaction.
Where applicable, experiments conform with Society ethical requirements