Polyurethane acrylates as effective substrates for sustained in vitro culture of human myotubes

Muscular disease has debilitating effects with severe damage leading to death. Our knowledge of muscle biology, disease and treatment is largely derived from non-human cell models, even though non-human cells are known to differ from human cells in their biochemical responses. Attempts to develop highly sought after in vitro human cell models have been plagued by early cell delamination and difficulties in achieving human myotube culture in vitro. In this work, we developed polyurethane acrylate (PUA) materials to support long-term in vitro culture of human skeletal muscle tissue. Using a constant base with modulated crosslink density we were able to vary the material modulus while keeping surface chemistry and roughness constant. While previous studies have focused on materials that mimic soft muscle tissue with stiffness ca. 12 kPa, we investigated materials with tendon-like surface moduli in the higher 150 MPa to 2.4 GPa range, which has remained unexplored. We found that PUA of an optimal modulus within this range can support human myoblast proliferation, terminal differentiation and sustenance beyond 35 days, without use of any extracellular protein coating. Results show that PUA materials can serve as effective substrates for successful development of human skeletal muscle cell models and are suitable for long-term in vitro studies. Statement of Significance We developed polyurethane acrylates (PUA) to modulate the human skeletal muscle cell growth and maturation in vitro by controlling surface chemistry, morphology and tuning material’s stiffness. PUA was able to maintain muscle cell viability for over a month without any detectable signs of material degradation. The best performing PUA prevented premature cell detachment from the substrate which often hampered long-term muscle cell studies. It also supported muscle cell maturation up to the late stages of differentiation. The significance of these findings lies in the possibility to advance studies on muscle cell biology, disease and therapy by using human muscle cells instead of relying on the widely used animal-based in vitro models.