Three dimensional engineered culture systems are powerful tools to rapidly expand our knowledge of human biology and identify novel therapeutic targets for disease. Bioengineered skeletal muscle has been recently shown to recapitulate many features of native muscle biology. However, current skeletal muscle bioengineering approaches require large numbers of cells, reagents and labour, limiting their potential for high-throughput studies. Herein, we use a miniaturized 96-well micro-muscle platform to facilitate semiautomated tissue formation, culture and analysis of human skeletal micro muscles (hµMs). Utilising an iterative screening approach we define a serum-free differentiation protocol that drives rapid, directed differentiation of human myoblast to skeletal myofibres. The resulting hµMs comprised organised bundles of striated and functional myofibres, which respond appropriately to electrical stimulation. Additionally, we developed an optogenetic approach to chronically stimulate hµM to recapitulate known features of exercise training including myofibre hypertrophy and increased expression of metabolic proteins. Taken together, our miniaturized approach provides a new platform to enable high-throughput studies of human skeletal muscle biology and exercise physiology.
An automatic method is established for layer-by-layer (LbL) assembly of biomimetic coatings in cell culture microplates using a commercial liquid-handling robot. Highly homogeneous thin films are formed at the bottom of each microwell. The LbL film-coated microplates are compatible with common cellular assays, using microplate readers and automated microscopes. Cellular adhesion is screened on crosslinked and peptide-functionalized LbL films and stem cell differentiation in response to increasing doses of bone morphogenetic proteins (2, 4, 7, 9). This method paves the way for future applications of LbL films in cell-based assays for regenerative medicine and high-throughput drug screening.
While the chemical signals guiding neuronal migration and axon elongation have been extensively studied, the influence of mechanical cues on these processes remains poorly studied in vivo. Here, we investigate how mechanical forces exerted by surrounding tissues steer neuronal movements and axon extension during the morphogenesis of the olfactory placode in zebrafish. We mainly focus on the mechanical contribution of the adjacent eye tissue, which develops underneath the placode through extensive evagination and invagination movements. Using quantitative analysis of cell movements and biomechanical manipulations, we show that the developing eye exerts lateral traction forces on the olfactory placode through extracellular matrix, mediating proper morphogenetic movements and axon extension within the placode. Our data shed new light on the key participation of intertissue mechanical interactions in the sculpting of neuronal circuits.
In zebrafish embryos, eye morphogenesis steers neuronal movements and axon extension in the overlying olfactory placode by exerting traction forces in the lateral direction, transmitted to the placode through extracellular matrix.• The cell bodies of olfactory placode neurons and cells in the adjacent eye tissue undergo highly correlated morphogenetic movements in the mediolateral direction, suggesting an interaction between the two tissues.• In mutants lacking eyes, mechanical stress at the lateral border of the placode and lateral movements of olfactory placode neurons are reduced, resulting in thinner placodes and shorter axons.• Enzymatic degradation of the extracellular matrix perturbs cell movements in the placode and decreases their correlation with eye cell movements, providing evidence that the matrix mechanically couples the two tissues.
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