CFD characterization of C2C12 dynamic culture on plant-derived scaffolds under intermittent flow for cultivated meat production

• Intermittent stirring promotes C2C12 growth and myogenesis on scaffold. • CFD reveals elevated velocity, shear stress, and improved uniformity at 40 rpm. • The CFD-DEM model could track scaffold velocity in a spinner flask. • CFD-guided dynamic culture identifies scale-up target conditions. Dynamic Culture is a crucial step in scaling up cultivated meat (CM) production, requiring the optimization of hydrodynamic flow, cell behavior, and scaffold suspension. Unlike the widely used microcarriers, the application of macro-porous scaffolds in spinner flask dynamic cultures remains largely unexplored, primarily due to their sensitivity to shear forces. In this study, intermittent stirring rather than continuous agitation was applied to preserve the structural integrity of the macro-porous decellularized plant scaffolds (DPS). The effects of intermittent stirring at 20 and 40 rpm on the proliferation and differentiation of murine myoblasts (C2C12) were evaluated. In parallel, computational fluid dynamics (CFD) and discrete element method (DEM) simulations were used to characterize fluid flow, shear stress distribution, and scaffold motion. Results showed that intermittent stirring at 40 rpm significantly enhanced C2C12 proliferation, formation of aligned fiber structures on DPS, and myogenic differentiation, as evidenced by the upregulation of myogenin ( Myog ), myosin heavy polypeptide 1 ( Myh1 ), and myosin heavy polypeptide 4 ( Myh4 ). Among the tested conditions, 40 rpm was associated with higher CFD-derived volume-average (VA) velocity magnitude, VA shear stress and VA energy dissipation rate. CFD-DEM simulations further revealed increased scaffold circulation velocity, suggesting improved nutrient exchange with the culture medium. This study demonstrates the potential of intermittent stirring as a scalable strategy for dynamic culture using DPS-like macro-porous scaffolds and provides hydromechanical insights relevant to CM production.