Universal Pressure-Loading Device for Live-Cell Imaging under Sustained Physiological Mechanical Stress

This protocol describes a hydrostatic pressure-loading device that facilitates real-time microscopic observation of adherent cells during sustained hydrostatic pressure stimulation, and is compatible with 3.5 cm commercial cell culture dishes. The apparatus consists of an airtight culture chamber fabricated from an aluminum base, an optically transparent poly(methyl methacrylate) cover, gas inlet/outlet ports integrated into the cover, and a sealed observation window. By connecting to a regulated gas source, the device maintains a stable hydrostatic pressure (0-200 kPa, adjustable) while enabling continuous phase-contrast or fluorescence imaging. Using this pressure-loading device, pressure-induced dose-dependent effects on cell phenotype and behaviors, such as morphology, proliferation, and migration, can be recorded. Furthermore, fluorescent signals can also be recorded in real time. Here, pressure-triggered Ca^2⁺ signaling heterogeneity and dynamics in breast cancer MDA-MB-231 cells and cervical cancer HeLa cells were observed and quantified by inverted fluorescence microscopy using time-lapse imaging. This platform integrates mechanical loading with live‑cell imaging to overcome limitations of conventional endpoint systems, providing a universal tool for mechanobiological studies.