Development and characterisation of a novel myotube-motoneuron 3D co-culture system

The aim of this Thesis was to characterise the behaviour and interaction of primary muscle derived cells (MDCs) and motoneurons within a collagen-based 3D in vitro culture system. Cells cultured under uniaxial tension within 3D collagen matrices are known to selforientate along the lines of principle strain. In the case of skeletal muscle cells, this leads to the formation of aligned myotubes, thereby generating cultures which more closely recapitulate the architecture of in vivo muscle. Since maturation of muscle in vivo is dependent on functional innervation, integration of this model with a physiologically correct neural input would further improve both the accuracy and complexity of the in vitro construct. Furthermore, reliable neuromuscular junction formation in 3D culture could have substantial benefits for the study of neuromuscular disease and the testing of novel therapeutic agents. The behaviour of primary rat MDCs within an established collagen-based 3D culture system was optimised and subsequently characterised. A comparison of this model to conventional 2D cell culture techniques was carried out using immunohistochemical and PCR analysis. Investigation of myogenin expression levels over a three week culture period in both 2D and 3D found no significant differences between the two systems, indicating a conserved ability for MDC differentiation in both models. Immunohistochemical data illustrated the alignment of uniaxial myotubes in 3D compared with randomly orientated and branched myotubes in conventional culture, demonstrating the improved biomimicity of myotubes developed in 3D and under directional tension. The presence of motoneurons within the 3D co-culture was found to promote maturation of the MDCs as indicated by levels of macroscopic construct contraction and by quantitative PCR analysis. Co-localisation of pre- and post- synaptic markers in culture indicated the presence of putative synaptic contacts within the model. The model presented in this Thesis represents a step forward in the development of physiologically accurate in vitro models of skeletal muscle, which may help in future investigations of skeletal muscle development, physiology and pathology