We developed a vertically integrated platform that combines genetically encoded reporters for cell-cycle phases and cell structure, photopatterned extracellular matrix islands of defined size, and long-term fluorescence imaging of proliferative and structural dynamics. Together with the automated Fab2Mic pipeline, this enables high-throughput tracking of cell migration and cell-cycle behavior under planar confinement.Using HT1080 fibrosarcoma cells, we found that planar confinement altered cell-cycle phase distributions and increased abnormal events, including prolonged G1 and mitotic slippage.

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We established an HT1080 fibrosarcoma four-reporter reference line that concurrently captures cell-cycle state and migration-associated cytoskeletal organization. To generate this line, HT1080 cells expressing EGFP-LifeAct were edited at the endogenous TUBA1B locus with a red fluorescent protein (RFP) tag via CRISPR/Cas9 editing, followed by lentiviral delivery of the FUCCIplex cassette under the EF1α promoter

Fab2Mic is a correlative fabrication-to-microscopy pipeline designed to eliminate the trial-and-error mapping of fabricated arrays to imaging fields of view (FOVs). By directly translating photofabrication layouts into microscopy coordinates, it enables accurate, efficient, and reproducible alignment between engineered patterns and live imaging. This reduces acquisition errors, saves imaging time, and improves the reliability of downstream analysis.

Long-term live-cell imaging under planar ECM confinement revealed differences in cell-cycle progression. Whereas cells in free space predominantly exhibited normal cell-cycle progression and division, confined cells showed an increased frequency of aberrant events. Together, these findings indicate that engineered planar confinement can directly modulate both cell morphology and the fidelity of cell-cycle progression.