Impact of 3-D Confinement on the Migratory Behavior of Young and Aged Cells: Implications for Cellular Senescence and Age-Related Decline

Cell migration is vital for development, wound healing, and tissue regeneration. Unchecked motility can trigger pathophysiological events, such as cancer metastasis. The prevailing notion is that cell motility reduces with aging. Most such studies use two-dimensional surfaces. Yet, in vivo, cells move through narrow, three-dimensional paths like micropores and channels. Confinement induces physical stress on cells, reshaping migration mechanisms. To date, the effects of confinement on aged and young cell migration are still unclear.

To compare young and aged cell motility in confinement, we fabricated PDMS-based microchannel devices using multilayer photolithography. The device had parallel 2D-like channels with perpendicular microchannels. Microchannels varied in width and height, keeping a fixed length (L = 200 µm). Microchannel dimensions were approximately 10x10 µm², 10x3 µm², 3x10 µm², and 10x6 µm². For each trial, early (10-15 passages) and late (>25 passages) dermal fibroblast suspensions were added to a larger channel's inlet for pressure-driven flow. Devices incubated for 10 mins at 37°C, letting cells adhere near microchannel openings. Media was added, and devices placed under a Nikon Ti2 Inverted Microscope with a Tokai Stage-Top Incubator. Experiments lasted 16 hours, imaging every 10 mins. MTrackJ tracked cell movement, calculating cell entry, persistence, velocity, and speed in microchannels. Experiments were repeated thrice and significance was determined using appropriate statistical tests (e.g., Student's t-test, ANOVA, Tukey's test).

Cells with 10-15 passages were deemed young, while those with >25 passages were classified as old due to halted division and elevated senescence-associated beta-galactosidase levels. Experiments showed young cells had a significantly reduced speed and velocity with higher confinement, with no significant change in cell entry among devices. Adding a chemotactic gradient for young cells to boost confined migration affirmed speed and velocity decline with increased confinement.

In the weeks leading up to the conference, we will analyze the effects of confinement on the migration of old cells. After establishing baseline cell motility for young and old cells, we will delve into how key migration factors like myosin II, actin, focal adhesion, and nuclear hardening influence old vs. young cell motility in confinement. Our objective is to manipulate pathways for rejuvenating aged cells to emulate youthful behavior. This research targets alleviating cellular senescence's negative effects on function, offering potential solutions for age-related cellular decline.