Intracellular Crowding Alters Cell Mechanics and Dynamics during Aging

Aging drives profound physical changes in cells, yet the underlying mechanisms remain unclear. In particular, the cytoplasm provides the essential physical environment for intracellular processes, but how its properties evolve with age is not well understood. Using fibroblasts derived from well-established mouse aging models, we investigate how cellular morphology, dynamics, and the physical state of the cytoplasm change with aging. Aged cells exhibit larger and rounder spreading profiles and migrate more slowly compared to young cells. Through particle fluctuations, optical tweezers, and force spectrum microscopy, we find that aging increases cytoplasmic stiffness and suppresses intracellular motion, even as active intracellular forces become stronger. Tomographic phase microscopy further reveals that aged cells possess a higher refractive index, indicating a denser, more crowded cytoplasm. This intracellular crowding underlies both the increased stiffness and the reduced internal dynamics, linking the mechanical and dynamic phenotypes of aging cells to fundamental changes in their cytoplasmic environment. Altogether, our results identify intracellular crowding as a distinctly physical phenotype underlying the altered mechanical and dynamical behavior of cells from aged organisms. Understanding how the physical nature of the cytoplasmic environment changes with aging may provide further insight into cellular dysfunction and future strategies to mitigate aging-related decline.