Live Cell Imaging during Mechanical Stretch

Summary

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Abstract

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

Introduction

Cells are subjected to mechanical loads in many tissues, and this mechanical stimulation has been shown to promote changes in patterns of gene expression, release of growth factors, cytokines, or remodeling of the extracellular matrix and cytoskeleton^1-4. The intracellular signals transduced from such mechanical stimuli occur through the process of mechanotransduction^5-7. In the respiratory system, one outcome of mechanotransduction is the increase in reactive oxygen species (ROS)^8,9 and pro-inflammatory cytokines¹⁰ in pulmonary epithelial cells in the presence of cyclic tensile strain. Strong evidence also suggests that exc....

Protocol

1. Construction of Membrane with Well Walls for Retention of Cell Culture Media (see Figure 1D for the final product)

1. Using polydimethylsiloxane (PDMS) sheets coated with Collagen I, cut the outline of the flexible membrane with a scalpel or a die.
2. Place each membrane in a 60 mm Petri dish for storage.
3. Creation of walls:
   1. Mix PDMS at a 10:1 weight ratio of elastomer A to elastomer B (curing agent).
   2. Pour 5 ml of fully mixed PDMS into 50 ml tubes.
   3. Place 50 ml tubes with uncured PDMS horizontally in a hybridization oven.
   4. Use the rotor function to coat the inner walls of the tubes at 8 rpm during ....

Results

Reactive Oxygen Species and Deformation

Previous studies have shown increases in reactive oxygen species (ROS) in airway and alveolar epithelial cells in response to cyclic stretch²¹. Reactive oxygen species include molecules and free radicals derived from molecular oxygen with high reactivity to lipids, proteins, polysaccharides, and nucleic acids^22-24. ROS serve as a common intracellular signal to regulate ion channel function, protein kinase/phosphatase activa.......

Discussion

A unique device for live cell imaging during mechanical stretch was developed; and this device was used in a protocol to study lung epithelial cell mechanobiology. In preliminary studies, it was found that a single held stretch stimulated the production of mitochondrial superoxide in bronchial epithelial cells. In addition, it was demonstrated that increased levels of mechanical strain caused direct damage to the integrity of a monolayer of alveolar epithelial cells.

To conduct these prelimina.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SmartMotor NEMA 34: 3400 Series	MOOG Animatics	SM3416D	Integrated motor, controller, amplifier, encoder and communications bus
Flexcell Membrane (Collagen I coated)	Flexcell International Corp	SM2-1010C	3.5" x 5.25" x 0.020"
Sylgard 184	Dow Corning Corporation		10:1
Hoechst 33342	Sigma-Aldrich	H1399	DAPI stain
MitoSOX	Sigma-Aldrich	M36008
Tiron	Sigma-Aldrich	D7389	mitochondrial superoxide label
DMEM			superoxide inhibitor
FBS
HEPES
50 ml tubes	Fisher Scientific	06-443-19	Any centriguge tube can be used to create an area for imaging.
Hybridization oven	Bellco Glass
MLE12 Cells	ATCC	CRL-2110	Mouse Lung Epithelial Cells
16HBE cells	ATCC	CRL-2741	Human Bronchial Epithelial Cells
AFM Indentation Experiments
Cantilever Beams for Nano-indentation	Budget Sensors	Si-Ni30
AFM	Asylum Research	MFP3D
Olympus microscope	Olympus	IX-71	Inverted microscope with 20X and 40X objectives.
AFM Leg Extenders	Asylum Research	Not available	AFM microscope
Finite Element Analyses
ABAQUS	Simulia	6.12
Software
ImageJ	NIH
Microscopes
Digital microscope	Life Technologies	EVOS XL Core	Initially a self standing company, now owned by Life Technologies.
Confocal microscope	Zeiss	LSM 710	2-photon upright microscope