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 cells⁵ and alveolar epithelial cells⁶ also undergo sustained mechanical stresses from tension and matrix stiffness-derived pressure.

There is growing evidence that confirms that mechanical pressure acts as a potent regulator of cell phenotype and behavior^7,8. In non-malignant cells, compressive stress enhances the proliferation of periodontal stem cells and induces osteoclast fusion through invasive protrusions⁹. Conversely, in cancer models, compression exhibits janus effects: it suppresses melanoma cell proliferation and migration while concurrently activating transcriptomic reprogramming that promotes chemoresistance^10,11. Furthermore, volumetric compression caused by hyperosmotic pressure can induce intracellular crowding and stimulate the redistribution of protein concentration and ionic strength via liquid-liquid phase separation (LLPS), which profoundly alters cell behaviors and fate. This physical perturbation enhances expansion of intestinal organoids through activated Wnt/β-catenin signaling and increased self-renewal of intestinal stem cells¹². In addition, in the cell nucleus, LLPS induced by volume changes is involved in gene transcriptional control through the coordination of transcription initiation, elongation, and termination^13,14.

To quantitatively investigate these pressure-induced biological effects, experimental systems capable of applying well-defined and homogeneous compressive stimuli are essential. For cells cultured in vitro, which are surrounded by an aqueous medium, hydrostatic pressure serves as a primary and physiologically relevant means of applying such compressive forces, which has been widely adopted to study cellular mechanotransduction¹⁵. Hydrostatic pressure can increase alkaline phosphatase activity and accelerate osteogenesis of human adipose-derived mesenchymal stem cells without supplementation of osteoinductive factors¹⁶. In non-alcoholic fatty liver disease, mechanical forces like hydrostatic pressure can contribute to portal hypertension without significant fibrosis, underscoring its role in tissue diversity¹⁷. Although hydrostatic pressure plays an important role in pathophysiological regulation, mechanistic studies of hydrostatic pressure face a critical limitation: conventional pressure chambers preclude real-time observation of live cells during stimulation. Most experimental systems only permit endpoint analysis, masking spatial and temporal dynamics of mechanotransduction events, such as Piezo1 mechano-activation, Ca²⁺ flux heterogeneity, and cytoskeletal reorganization. This gap limited the understanding of how hydrostatic pressure drives the diversity of phenotypes in tumors or other tissues.

To address this technological gap, a novel experimental platform was developed that enables real-time observation of cellular responses to precisely controlled hydrostatic pressure through optical imaging. This system integrates three key capabilities: (1) application of sustained hydrostatic pressure within a range of 0–100 kPa via an adjustable gas-pressure controller (The pressure gauge has a range of 0–200 kPa, and the applied pressures can be selected based on physiological parameters. For instance, based on the reported compressive stresses of various cell types: 8-16 kPa for human bone marrow stromal cells¹⁸, 12–24 kPa for vascular smooth muscle cells¹⁹, and 0–15 kPa for human mammary epithelial cells²⁰.); (2) continuous live-cell imaging with high temporal resolution during hydrostatic pressure application; and (3) maintenance of physiological culture conditions throughout extended experiments. This approach allows for direct visualization of dynamic processes, including Piezo1 channel recruitment and activation, calcium signaling waves, and cytoskeletal remodeling that occur in response to mechanical stimulation. In addition, this device is compatible with most commercially available culture dishes and inverted microscopes, making it highly convenient to use in laboratories. The ability to monitor these intracellular biochemical events in real time provides unprecedented insights into how cells sense and adapt to hydrostatic pressure. By capturing the temporal sequence of mechanotransduction events, which include the initial sensing of force, downstream signaling, and phenotypic changes, this platform provides a powerful tool for investigating the fundamental mechanisms by which hydrostatic pressure influences cell behavior in physiological and pathological contexts.

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-antimycotic solution, and a calcium imaging probe for cell culture and Ca²⁺ labeling.

2. Device preparation

1. Manufacture
   1. Custom-design the central component of this study. Precision-machine the aluminum base and PMMA cover according to the specifications (See Figure 1 and Supplementary Figure 1).
   2. Cut the silicone gasket, and process it to the required size.
      ​NOTE: The optical transmittance of PMMA material is sufficient for standard transmitted light imaging. All components were sterilizable and biocompatible for subsequent cell culture experiments. No special requirements are imposed on the manufacturing tolerances of this chamber (a tolerance of ±0.1 mm was adopted in this study).
2. Sterilization
   1. To sterilize the base (Aluminum), autoclave it at 121 °C for 20 min.
   2. To sterilize the cover (PMMA), wipe it with 75% ethanol, then UV sterilize for 30 min.
   3. Sterilize the silicone gasket by autoclaving at 121 °C for 20 min.

3. Cell processing

1. Cell culture
   1. Seed cells in 35 mm dishes at 30%–70% confluency. Culture the cells for 24 h in RPMI-1640 or L15 medium (supplemented with 10% NCS) in a CO₂ incubator at 37 °C with 5% CO₂.
   2. For cell passaging, dissociate the cells with 0.25% trypsin and terminate digestion with complete medium. Use the cells for reseeding once they reach 70%–80% confluence.
2. Ca²⁺ staining
   1. Remove the medium, and then wash the cells twice with pre-warmed Hank's Balanced Salt Solution (HBSS) in a biosafety cabinet.
   2. Stain the intercellular Ca²⁺ with 5 µM calcium imaging probe in serum-free medium. Incubate the cells for 20 min at 37 °C in a light-protected environment.
   3. Remove the dye, wash the cells three times with HBSS, and then de-esterify for 30 min in serum-free medium.

4. Device assembly (Figure 2)

1. Transfer the culture dish to the central window of the pressure chamber, then remove the culture medium.
2. Place the sterilized gasket in the culture dish.
3. Align the cover over the dish-gasket stack.
4. Tighten the screws diagonally by first hand-tightening them until resistance is felt, then adding 1.5 additional turns using a torque-controlled driver.
5. Connect the silicone gas tube to the Luer-lock port and inject mixed gas into the device to check for leakages.
6. Disconnect the silicone gas tube and inject fresh medium into the cell culture dish through the Luer-lock port using a 5 mL sterile syringe.

5. Pressure loading and imaging

1. Connect silicone gas tubing (inside diameter 4 mm) from the Luer-lock port on the silicone gasket to the digital pressure gauge and the gas tank.
2. Mount the assembled device on the microscope stage.
3. Calibrate the pressure.
   1. Validate the stability of the 0–200 kPa range using the gauge.
      ​NOTE: A mixed gas containing 75% nitrogen, 20% oxygen, and 5% carbon dioxide is used for HeLa cell culture. A mixed gas containing 80% nitrogen and 20% oxygen is used for MDA-MB-231 cell culture. The type of gas used in the pressure-loading experiment can be replaced according to the cell type, the medium type, or specific experimental requirements. No components are model-specific, and the dimensions of the pressure detection module and its inlet tubing can be selected as required.
4. Set the target pressure (e.g., 100 kPa) via the digital pressure regulator.
5. Initiate imaging once the target pressure is reached.
6. Imaging
   1. Adjust the stage, confirm the imaging area, and ensure that the cells are in focus.
   2. Acquire fluorescence images of stained cells using an inverted fluorescence microscope equipped with a 20× objective, with the following parameters: 400 ms exposure time, 5 s time-lapse intervals, and a total imaging duration of 10 min (FITC filter: Ex/Em = 490/525 nm, cutoff = 515 nm).
   3. Image under non‑pressurized conditions for 10 min to ensure the proper operation of the system and to obtain baseline signal data.
   4. After opening the gas valve to load pressure, readjust the stage height to ensure the cells are in focus, then start imaging dynamic calcium signals.
   5. Select the objective lens based on the bottom thickness of the culture dish and the objective's working distance. Choose the exposure time used to enable monitoring of calcium signal oscillations while avoiding excessive photobleaching and photodamage.
   6. Adjust parameters according to the probe type and experimental requirements when observing other dynamic signals.

6. Troubleshooting

1. Refer to Table 1 for troubleshooting advice.

7. Statistical analysis

1. Present all data as mean ± standard deviation (SD). GraphPad Prism software (USA) was used to compare results between two groups by the unpaired Student's t-test. Consider differences as statistically significant when **p < 0.01, ***p < 0.001, or ****p < 0.0001.

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