Microfluidic Mechanical Reactivation of Aged Stem Cells

ABSTRACT Stem cell aging significantly impairs therapeutic efficacy, requiring innovative strategies to restore potency. We present a microfluidic cell-compressing platform for reactivation (μ-CPR) designed to apply controlled hydrodynamic deformation to late-passage stem cells. This mechanical stimulation facilitates functional reactivation without external chemical cues. Within a defined window, μ-CPR effectively reduces oxidative stress, SA-β-Gal activity, and γH2AX foci, while simultaneously restoring proliferation and canonical stemness markers (OCT4, SOX2, and KLF4). Mechanical stimulation via μ-CPR induces coordinated structural remodeling: nuclei become more compact, actin cortex organization is restored, α-actinin redistributes to focal adhesions, and microtubule networks are restructured, suggesting a rebalanced intracellular tension. Transcriptomic and proteomic analyses reveal that this process reprograms extracellular matrix remodeling and DNA repair pathways while attenuating pro-fibrotic and senescence-associated secretory phenotype (SASP)-associated pathways. Crucially, this reactivation occurs without compromising fundamental MSC hallmarks, preserving intrinsic immunophenotypes and multilineage differentiation potential. Functionally, μ-CPR-processed stem cells demonstrate restored in vitro wound closure and enhanced tissue repair in vivo , with efficacy appearing dependent on mechanical dosage. This platform establishes a non-genetic, mechanobiological approach to restoring stem cell function, offering a scalable strategy for functional reactivation and potentially paving the way toward comprehensive cellular rejuvenation.