High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition

Summary

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-β dependent increase in contractility during the epithelial-to-mesenchymal transition.

Abstract

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications, however, require direct quantification of these forces.

We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.

In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-β, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-β exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

Introduction

Acto-myosin contractility is an essential element of active cell mechanics, impacting cell behaviors from motility and proliferation to stem cell differentiation. In tissues, contractility drives activity from polar separation in embryogenesis, to airway constriction and cardiac activity. Critically, to generate tension, cells must first adhere to their extracellular environment. In doing so, this contractility generates traction forces on their surroundings. Traction Force Microscopy (TFM) has emerged in a multitude of forms as a way to quantify these forces from diverse cells under different conditions.

The field of TFM has seen an except....

Protocol

NOTE: The following protocol will guide researchers in fabricating and using the multi-well TFM dish shown in Figure 1.

1. Preparation of PDMS silicone substrates

1. Preparation of PDMS silicone rubber mixture based on a composite mixture of two commercially available kits.
   1. Add Part A and Part B of PDMS kit (e.g., GEL-8100, see the Table of Materials) in a 1:1 weight ratio into the 50 mL tube.
      NOTE: The mixture is mixed on a rotator at a speed slow enough for the mixture to flow back and forth during revolution to ensure complete mixing.
   2. Add the required ....

Results

Before addition of TGF-β, a confluent monolayer of cells has a cobblestone like shape and is tightly packed. Upon TGF-β treatment, cells become more elongated in morphology, enlarging the cell area and acquiring a more mesenchymal phenotype. Utilizing the multi-well device fabricated with soft PDMS elastomers, the physical properties of cells in a total 17 different conditions were studied. The cells were treated with four different TGF-β concentrations (0.5, 1, 2, and 4 ng.......

Discussion

For the success of this method, it is critical to have a uniformly coated sample with a constant thickness of approximately 100 µm. The modulus should be carefully chosen to examine the physical significance of the biological system of interest. When fabricating a top layer, the concentration of the fiducial fluorescent particles should be optimized for accurate analysis of displacement and traction stress. Analyzing isolated single cells requires a denser fiduciary layer than measuring confluent monolayers. Additio.......

Materials

Name	Company	Catalog Number	Comments
Plate
GEL-8100	Nusil Technology	GEL-8100	High Purity Dielectric, Soft Silicone Gel kit
Dow Corning Sylgard 184 Silicone Encapsulant Clear 0.5 kg Kit	Ellsworth Adhesives	184 SIL ELAST KIT 0.5KG	curing agent
Custom Cut Glass	Hausser Scientific Company		109.6mm± x 72.8mm± x 1mm thickness
Target 2TM Nylon Syringe Filter	ThermoFisher Scientific	F2513-4
96-well Stripwell Egg Crate Strip Holder	Corning	2572
Polystyrene Universal Microplate Lid With Corner Notch	Corning	3099
Ethyl alcohol	Greenfield Global	P016EA95	0.95
2-Propanol	Sigma-Aldrich	190764	ACS reagent, ≥99.5%
Surface Coating
Sulfo-SANPAH Crosslinker	Proteochem	c1111-100mg
Fibronectin bovine plasma	Sigma-Aldrich	F1141-1MG	solution, sterile-filtered, BioReagent, suitable for cell culture
PBS, 1X	Wisent	319-005-CL	pH 7.4, without calcium and magnesium
DMSO	Sigma-Aldrich	472301
Cell Culture
DMEM, 1X	Wisent	319-005-CL	4.5g/L glucose, with L-glutamine, sodium pyruvate and phenol red
FBS (Fetal Bovine Serum)	Wisent	080-150	Premium Quality, Endotoxin <1, Hemoglobin <25
HEPES	Wisent	330-050-EL	1M, free acid
Human Insulin Recombinant	Wisent	511-016-CM	USP grade
Penicillin-Streptomycin Solution	Wisent	450-201-EL	100 X, sterile filtered for cell culture
L-Glutamine solution	Wisent	609-065-EL	200mM solution, sterile filtered for cell culture
Amphotericine B	Wisent	450-105-QL	250μg/ml, sterile filtered for cell culture
Recombinant Human TGF-β1	Peprotech	100-21	HEK293 Derived
Acetic acid	Sigma-Aldrich	537020	Glacial, ≥99.85%
Cictric acid	Sigma-Aldrich	251275	ACS reagent, ≥99.5%
NMuMG	ATCC	CRL-1636	Mouse Mammary Gland Cell Line
Sodium azide	Fisher Schientific	AC190385000	99%, extra pure, ACROS Organics
Potassium hydroxide	Sigma-Aldrich	221473	ACS reagent, ≥85%, pellets
TritonX-100	Sigma-Aldrich	X100	laboratory grade
Bead Synthesis
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)	Sigma-Aldrich	468495-100MG	97%
Methyl methacrylate	Sigma-Aldrich	M55909-500ML	contains ≤30 ppm MEHQ as inhibitor, 99%
Inhibitor Remover	Sigma-Aldrich	306312-1EA	Prepacked column for removing hydroquinone and monomethyl ether hydroquinone
Methacryloxylpropyl Terminated Polydimethylsiloxane	Gelest	DMS-R31 (25,000g/mol)	Polydimethylsiloxane stabilizer, 25,000g/mol, 1,000 cSt
2,2′-Azobis(2-methylpropionitrile) (AIBN)	Sigma-Aldrich	441090-25G	98%
Hexane	Sigma-Aldrich	296090-2L	anhydrous, 95%
Hexane, mixture of isomers	Sigma-Aldrich	227064-1L	anhydrous, ≥99%
Whatman qualitative filter paper, Grade 1	Sigma-Aldrich	WHA1001055	circles, diam. 55 mm,
Equipment
Laurell WS-650Mz-23NPPB	Laurell Technologies
UVP Handheld UV Lamp Model UVGL-58	VWR	21474-622
Rheometer	Anton Paar	MCR 302 WESP