Myoblasts in 3D displayed actin stress fibers that were mainly oriented along the predominant longitudinal direction of gel and cell axis. Actin stress fibers were evenly distributed at the perinuclear regions. In addition, a reticulated network of thinner actin fibers was present at the extremities of myoblasts. 2D cultured myoblasts were randomly oriented in the culture plate but also displayed actin stress fibers that were mainly oriented along the longitudinal cell axis. However, myoblasts in 2D typically Madecassoside exhibited larger actin fibers at the cell periphery with rare and thinner actin fibers located at the supra-nuclear and subnuclear regions. There was no significant difference in the level of actin expression myoblasts. Interestingly, actin fibers thickened conditions upon myotube differentiation. Engineering skeletal muscle tissue fills a critical gap in the currently available physiological tools, between traditional 2D cell cultures and whole animal experiments, with an approach that places cultured cells in an environment that more closely reproduces the complex 3D structure of native tissue. Although the ECM surrounding individual myofibers in vivo consists mainly of collagen IV, laminin, and heparin sulphate-containing proteoglycans, there is large evidence to suggest that fibrin-based 3D Barlerin scaffold has great potential for applications in tissue engineering and regenerative medicine. Fibrin networks have been shown to support myocyte anchorage and the formation of the engineered cardiac and skeletal muscle tissue. However characterization of myocyte spreading and adhesions within the 3D environment and cell-scaffold interaction during engineered muscle tissue formation has not been studied before. In the present study, we show that human myoblasts within a fibrin scaffold spread and form actin stress fibers, even if the low strain modulus of fibrin alone would predict a round cell morphology and the absence of stress fibers. This apparent paradox is consistent with what has been previously reported in mouse NIH 3T3 fibroblasts embedded on fibrin gel and suggests that myoblasts, as fibroblasts or mesenchymal cells, sense and apply strains large enough to enter the strain-stiffening regime of fibrin elasticity. Moreover, our data indicate that the elastic modulus of the fibrin construct increased with the density of myoblasts, thereby indicating that myoblasts also actively stiffened the 3D fibrin.