Engineering 3D muscle microtissues by co-culturing human myoblasts and fibroblasts

摘要

Introduction: Actual in vitro models for skeletal muscle tissue engineering present many limitations for high content screening analysis: 2D models do not mimic the native cellular microenvironment, whereas 3D models require large amounts of cells, thus limiting the reproducibility of experiments'. In our lab, we developed a microfabricated platform for generating arrays of 3D microtissues with force tracking in real time. Here, we demonstrate how tissue formation, stability, and cell differentiation in microtissues generated from immortalized human myoblasts can be improved by co-culturing the myoblasts with fibroblasts. Materials and Methods: Human immortalized myoblasts were kindly provided by the Institute of Myology (Paris, France). Human fibroblasts BJ-5ta were obtained from ATCC. Polydimethylsiloxane (PDMS) platforms were made by casting Sylgard 184 liquid prepolymer over a silicon master fabricated by UV photolithography. The platforms consisted of 800x400x160μm wells containing two T-shape microcantilevers. A cooled suspension of cells within a reconstitution mixture consisting of collagen Ⅰ and matrigel was then added to the platform, resulting in around 200 cells per well, before incubation at 37°C to induce collagen/matrigel polymerization. Cell-generated forces were quantified from the deflection of the microcantilevers. Fig. 1: Engineering of human muscle microtissues. (A) Schematic of the microfabrication of the platform. (B) Temporal evolution of muscle microtissues constructed in collagen/matrigel gels in our microfabricated platform. The scale bar is 200μm. (C) Effect of human myoblasts/fibroblasts co-culture on the stability of microtissues after 3 days in culture. Results and discussion: After cell seeding, the collagen/matrigel matrix contained evenly distributed round cells. Over time, cells elongated and compacted the matrix to form a microtissue spanning between the two microcantilevers. After 3 days in culture, we observed that micromuscles composed only of human myoblasts tended to rupture (only 46% of stable tissues) whereas micromuscles consisting of 20% fibroblasts and 80% human myoblasts remained stable over time (94% of stable tissues). After 3 days in culture, the tension generated by muscle microtissues composed only of human myoblasts reached 20 μN while tissues composed of 20% fibroblasts and 80% human myoblasts generated a tension of 13 μN. We posit that fibroblasts helped stabilizing muscle microtissues by lowering the tissue-generated tension and by secreting additional extracellular matrix, thus reinforcing the integrity of the tissue. Interestingly, further analysis showed that fibroblasts improved spontaneous myoblasts differentiation toward myotubes after 5 days in culture. Conclusions: In this work we showed that engineered microtissues from a co-culture of human myoblasts and myoblasts exhibited a more stable morphology, thanks to a reinforcement of the extracellular matrix secreted by fibroblasts. The degree of differentiation of the muscle microtissues was also improved by the co-culture. The overall similarity of structural and functional characteristics between muscle microtissues and in vivo skeletal muscle is promising and our novel method for micromuscle generation opens the potential to high throughput, low volume screening applications. Moreover, the model's ability to quantitatively demonstrate the impact of biological and physical parameters on the formation, maturation, structure and function of muscle tissue provide unique opportunities to elucidate mechanisms of myogenesis in stable, three-dimensional, working muscle preparations. The authors thank the members of the technical staff of the PTA cleanroom in Grenoble for their technical support. We acknowledge the platform for immortalization of human cells from the Institute of Myology for providing us with human immortalized myoblasts. This work was supported by the European Commission (under FP7) via an ERC starting grant (BIOMIM, GA 259370) and by the Association Francaise contre les Myopathies (AFM-Telethon, project n°316530). CM is indebted to AFM-Telethon for providing a postdoctoral fellowship (n°16673).