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

## TL;DR

This paper introduces a new system to study how cell migration and cell cycle interact under spatial constraints, revealing changes in cell behavior and cycle progression.

## Contribution

The novel contribution is a vertically integrated platform combining fluorescent reporters, photopatterned matrices, and automated imaging for live-cell tracking under confinement.

## Key findings

- Planar confinement reduces cell area and cytoskeletal spread while altering cell cycle phase distributions.
- Confined conditions increase abnormal cell cycle events like prolonged G1 and mitotic slippage.
- Cell migration is faster during the G1 phase of the cell cycle under confinement.

## Abstract

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.