A vertically integrated system for tracking and assessing cell-cycle-aware phenotypes under confinement.

Quantitative cell biology often examines migration and cell-cycle (CC) progression separately, limiting insights into their interplay under spatial constraints. Here, we present a vertically integrated platform combining multiplexed fluorescent reporters for CC phases, actin, and tubulin with photopatterned extracellular matrix islands of defined sizes, alongside an automated imaging pipeline (Fab2Mic) for high-throughput, live-cell tracking of migration and CC dynamics under planar confinement. Using HT1080 fibrosarcoma cells, we observed that planar confinement progressively reduced cell area and cytoskeletal spread, altered CC phase distributions, and increased abnormal CC events, including prolonged G1 and mitotic slippage, which is unique to confined conditions. Dynamic imaging revealed CC-dependent motility variations, with faster migration in G1. This system enables systematic, CC-aware mechanobiology studies under controlled confinement, providing access to dynamic phenotypes inaccessible to static assays and offering a scalable approach for mechanistic investigations and screening applications.