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

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