Ultrasound-mediated extracellular matrix-preserving detachment suppresses extrinsic aging of mesenchymal stromal cells

Large-scale expansion of mesenchymal stromal cells (MSCs) is essential for regenerative medicine. However, serial passaging is accompanied by progressive cellular aging and deterioration in cell quality. Conventional enzymatic passaging may accelerate this process by adding non-physiological stress beyond intrinsic replicative senescence. We developed an ultrasound-mediated, enzyme-free passaging system that enables complete and gentle detachment of MSCs while preserving the extracellular matrix and adhesion-associated structures. Ultrasound-mediated passaging maintained high cell viability and sustained proliferation over extended culture periods, resulting in higher cumulative cell yields than trypsin-based passaging. MSCs cultured under enzyme-free conditions preserved their spindle-shaped morphology, suppressed cell hypertrophy, and exhibited attenuated senescence-associated features, including reduced senescence-associated β-galactosidase activity and attenuated p53 induction. Ultrasound-mediated cell passaging did not compromise self-renewal capacity, differentiation potential, or the expression of stemness-associated markers compared with that induced by conventional trypsinization. Thus, passage-associated aging is predominantly due to procedure-induced extrinsic stress rather than intrinsic replicative limits.