Droplet Microfluidics-Enabled Mitochondrial Transfer from Young to Senescent MSCs to Ameliorate Cellular Senescence

Mesenchymal stem cells (MSCs) are widely used for tissue repair and regeneration, but prolonged in vitro expansion induces senescence and limits their therapeutic efficacy. Given the key role of mitochondria in cellular senescence and metabolic regulation, mitochondrial transfer may offer a promising strategy for ameliorating senescence-associated phenotypes. However, conventional mitochondrial transfer methods, such as coculture and microinjection, are limited by poor quantitative control, low throughput, and potential cell damage. Here, an inertial-focusing-assisted droplet microfluidic platform was developed for high-throughput, high-efficiency, and quantitative control of mitochondrial transfer at the single-cell level. The platform achieved 29.2% single-cell droplets and 1.3% multicell droplets, with a transfer efficiency of up to 56% at a droplet generation rate exceeding 4000 Hz. Using this platform, the transfer of 19 mitochondria from young adipose-derived MSCs (ADSCs) to senescent ADSCs enhanced proliferation capacity and metabolic activities, reduced senescence-associated markers, and transformed the senescent phenotype into a young MSC-like phenotype. The developed technique provides a cell therapy strategy for mitochondrial-related diseases.