Microfluidic Mechanical Reactivation of Senescent 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 reduces oxidative stress, SA-β-gal activity, and γ-H2A.X foci, while enhancing proliferation and increasing the expression of canonical stemness-associated markers, including 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 reorganization of intracellular mechanical architecture. 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 stem cell hallmarks, preserving intrinsic immunophenotypes and multilineage differentiation potential. Functionally, µ-CPR-processed stem cells demonstrate enhanced in vitro wound closure and improved tissue repair in vivo, with efficacy dependent on the applied mechanical dose. This platform establishes a non-genetic, mechanobiological approach to functional stem cell reactivation, offering a scalable strategy for restoring stem cell function and providing a foundation for future cellular rejuvenation strategies.