High-throughput and label-free isolation of senescent murine mesenchymal stem cells

Abstract

Under internal or external insults such as aging and oxidative stresses, cells are induced into a senescent state and stop cellular division permanently. As senescent cells (SnCs) accumulate, the regeneration capacity of biological tissue would be compromised, which has been found to be associated with a plethora of age-related disorders. Therefore, isolating SnCs becomes necessary. To address the lack of effective surface markers for SnCs isolation, a label-free microfluidic device was proposed in this paper, in which a spiral microchannel was deployed to isolate SnCs based on their size differences. We adopted a well-received cellular senescence model by exerting excessive oxidative stress to murine mesenchymal stem cells. This model was then validated through a series of SnCs characterizations including size measurement, p16INK4a expression level, senescence-associated beta-galactosidase, and doubling time. The senescence chip demonstrated an efficiency of 75% and viability over 85% at a flow rate of 5 ml/min. The average cell size from the inner outlet was 5 μm larger than that from the outer outlet. The isolated cells had a sixfold higher p16INK4a expression level. Overall, the chip had an area under curve of 0.719 in the receiver operating characteristic analysis, showing decent performance in sorting SnCs. By having the ability to perform size-based sorting at a high flow rate, such a microfluidic device can provide high-throughput and label-free isolation of SnCs. To further improve the isolation performance, the device can be modified to introduce additional physical biomarkers of SnCs such as stiffness. This device poses a good potential in purification for cytotherapy or estimation of biological age.