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

Summary

The pressure-loading device described here enables time-lapse optical imaging of cultured cells and is compatible with standard cell culture dishes and inverted microscopes. Using this device, the tumor hydrostatic pressure microenvironment is confirmed to enhance calcium signal transduction, providing a feasible platform for studying mechanical signal transduction in the tumor microenvironment.

Abstract

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.

Introduction

Numerous cells, tissues, and organs experience sustained or intermittent compressive forces as part of their physiology¹. For instance, alveolar cells are subjected to cyclic pressurization during respiration², whereas bladder epithelial cells endure mechanical loads during urine retention³. In pathological contexts such as solid tumors, cancer cells are continually exposed to compressive forces originating from spatial confinement and elevated interstitial fluid pressure⁴. Similarly, in other diseases like meniscus degeneration and pulmonary fibrosis, meniscal cells

Protocol

1. Materials

1. Device specifications (See Figure 1)
   1. Prepare the components based on the specifications mentioned below:
      Base: aluminum (Ø 56 mm, height 15 mm), central window Ø 40 mm.
      Cover: PMMA (Ø 56 mm) with: 6 × M3 threaded holes, Luer-lock gas port, silicone gasket (inside diameter 25 mm, outside diameter 32 mm, thickness 0.8 mm)
      ​Pressure regulator: (0–200 kPa range)
2. Cells and reagents
   1. Use MDA-MB-231 and HeLa cells in this study. Use L-15 medium, RPMI 1640 medium, newborn calf serum (NCS), 1% antibiotic-....

Results

To verify the real-time imaging function of this hydrostatic pressure-loading device, the dynamics of calcium signals in tumor cells following the application of hydrostatic pressure were observed. Confluent MDA-MB-231, HeLa and A549 cells were seeded into 35 mm culture dishes for subsequent use. After being stained with a calcium imaging probe, the serum-free cultured cells were subjected to a hydrostatic pressure of 40 kPa. After 10 min of hydrostatic pressure loading, the total calcium level in cancer cells increased .......

Discussion

This protocol showed a universally adaptable pressure-loading device that enabled real-time observation of mechanotransduction dynamics under tunable hydrostatic pressure. By integrating an optically transparent PMMA cover with a precision-machined aluminum base, the system overcomes the critical limitation of conventional chambers, which can only perform endpoint analysis. This pressure-loading device, working with a standard inverted microscope, can provide dynamic information through time-lapse imaging. The compatibil.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibiotic-Antimycotic (100x)	Thermo Fisher Scientific, USA	15140122	1% concentration in complete medium
Autoclave	Panasonic, Japan	MLS-3751-PC	For sterilizing the aluminum base and silicone gasket
Biosafety Cabinet	Thermo Fisher Scientific, USA	1300 Series A2 Class II, Type A2 Biological Safety Cabinets	Provides a sterile environment for cell handling
Cal-520 AM, cell-permeant calcium indicator	AAT Bioquest, USA	21131	For dynamic detection of calcium ion concentration in living cells
Cell Culture Dish (35 mm)	BIOFIL, China	TCD010035	Standard 35 mm dish used for seeding cells
Chamber Base (Aluminum)	Quanyi, China	/	Custom-designed Ø 56 mm, H 15 mm, central window Ø 40 mm
Chamber Cover (PMMA)	Quanyi,  China	/	Custom-designed Ø 56 mm, with 6× M3 threaded holes, Luer-lock gas port
CO2 Incubator	Thermo Fisher Scientific, USA	HERAcell 3111	Maintains culture conditions at 37°C with or without 5% CO2
HBSS (Hanks' Balanced Salt Solution)	Beyotime, China	C0219	For washing cells before and after staining
HeLa cell line	Cell Resource Center, Institute of Basic Medical Sciences, CAMS/PUMC, China	CRM-CCL-2	Human cervical cancer cells
Inverted Fluorescence Microscope	Nikon, Japan	Eclipse Ti2	For fluorescence imaging
Leibovitz's L-15 Medium	Thermo Fisher Scientific, USA	41300039	For MDA-MB-231 cell culture
MDA-MB-231 cell line	Cell Resource Center, Institute of Basic Medical Sciences, CAMS/PUMC, China	CRM-HTB-26	Human breast cancer cells
Newborn Calf Serum (NCS)	Thermo Fisher Scientific, USA	16010142	10% concentration in complete medium
Pressure Regulator	Hongrun, China	DP20	Range: 0-200 kPa
RPMI 1640 Medium	Thermo Fisher Scientific, USA	31800022	For MDA-MB-231 cell culture
Sterile Syringe (5 mL)	KDL, China	1101010198	For injecting medium or reagents through the Luer-lock port
Trypsin (0.25%)	Thermo Fisher Scientific, USA	2520056	For dissociating adherent cells for passaging