Résumé :
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Rationale: Engineered muscle tissue (EMT) from human cells may provide advanced in vitro models for drug testing and for pathophysiological analysis of musculardisorders. 3D cultures allow cell-cell and cell-extracellular matrix (ECM) interactions that would mimic more closely the in vivo conditions, and allow measurements of thecontractile performance of engineered muscle tissue. Another advantage of the 3D culture system for muscle cells is the ability to keep differentiated cells in culture fora longer time than in classical 2D, where cells detach from contractions. However, a prerequisite for using muscle cells culture in 3D as a model is its characterization,including analysis of the proliferation and differentiation steps as well as cell-matrix interactions. Methods: We are using and characterizing a 3D system model thatconsists of human primary myoblasts embedded in a fibrin matrix casted between polydimethylsiloxane posts. The mechanical tension applied between the posts bythe coalescence of the gel and the cell tension stimulates alignment of the cells. Structural features of myoblasts and myotubes were determined by confocal andtransmission electron microscopy. Analyses of early differentiation steps were assessed by RT-qPCR and Western blot after up to 14 days of culture.Results: In our3D system, myoblasts proliferate, align and fuse to form parallel array of myotubes with regular striation pattern. The architecture of adhesion complexes in 3D wasremarkably different from those of myoblasts in 2D cultures. In addition, the nucleus shape was typically ellipsoid in 3D whereas spheroid nuclei were prominent in 2D(p<0.001). mRNA and protein expression of myogenin, alpha-actinin and myosin chains reveal a differentiation kinetic similar to the classical 2D cultures.Conclusionsand future directions: Our 3D cultures enable the in vitro reproduction of aligned myotubes replicating muscle tissue patterns and reveal 3D specific adhesion patterns.Our results support the goal of creating physiological relevant EMT from human biopsy. The full characterization of this system will allow its use in the study ofpathophysiological mechanisms, as well as the study of the cell-cell and cell-extracellular matrix interactions.
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