Jang, S. B.; Jeon, T.-I.; Kang, G. H.; Seo, D.; Kim, H.; Yeo, H.; Seok, J.; Lim, K. M.; Dayem, A. A.; Kim, S. J.; Song, K.; Kwak, Y.; Hur, J.; Chung, A. J.; Cho, S. G.

Stem cell aging significantly impairs therapeutic efficacy, requiring innovative strategies to restore potency. We present a microfluidic cell-compressing platform for reactivation (-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, -CPR effectively reduces oxidative stress, SA-{beta}-Gal activity, and {gamma}H2AX foci, while simultaneously restoring proliferation and canonical stemness markers (OCT4, SOX2, and KLF4). Mechanical stimulation via -CPR induces coordinated structural remodeling: nuclei become more compact, actin cortex organization is restored, -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, -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.