Many of the protocols in contemporary tissue engineering remain insufficiently optimised. Methodologies for culturing the complex structures of muscle tissue are particularly lacking, both in terms of quality and quantity of mature cells. Here, we analyse images from in vitro experimentation to quantify the effects of the composition of culture media on mouse-derived myoblast behaviour and myotube cell quality. We then apply computational modelling to predict the optimum range of media compositions for culturing. We define metrics of uniformity of myonuclei distribution as an early indicator of cell quality and difference in myonuclei density over time as an indicator of cell quantity. Analysis of live and static images of muscle cell differentiation revealed that changes in culture media result in significant changes in indicators of cell quantity and quality as well as changes in myoblast migratory behaviour. By describing media composition as a set of functions of cell behaviour we designed a model for predicting cell quality. Cell behaviours were taken directly from experimental images or inferred using Approximate Bayesian Computation and applied as inputs to an agent-based model of cell differentiation with cell quality indicators as outputs. Our results suggest that culturing muscle cells in a neural cell differentiation medium does not diminish cell quality. We show that, while high concentrations of serum are detrimental to cell development, increasing serum concentration raises the total amount of myoblast fusion, leading to a trade-off between the quantity and quality of cells produced when choosing a culture medium. Our numerical results provided a good prediction of experimental results for media with 5% serum provided the background cell proliferation rate was known.
The composition of fiber types within skeletal muscle impacts the tissue's physiological characteristics and susceptibility to disease and ageing. In vitro systems should therefore account for fiber type composition when modelling muscle conditions. To induce fiber specification in vitro, we designed a quantitative contractility assay based on optogenetics and particle image velocimetry. We submitted cultured myotubes to long-term intermittent light stimulation patterns and characterized their structural and functional adaptations. After several days of in vitro exercise, myotubes contract faster and are more resistant to fatigue. The enhanced contractile functionality was accompanied by advanced maturation such as increased width and upregulation of neuron receptor genes. We observed an upregulation in the expression of distinct myosin heavy chain isoforms (namely, neonatal-Myh8 and fast-Myh), which induced a shift towards a fast fiber phenotype. This long-term in vitro exercise strategy can be used to study fiber specification and refine muscle disease modelling.
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