Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Summary

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Abstract

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor β, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor α. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Introduction

Fibrosis is involved in the pathogenesis of various chronic progressive diseases, such as interstitial pulmonary disease, cardiac fibrosis, liver cirrhosis, terminal renal failure, systemic sclerosis, and autoimmune disease¹. Among interstitial lung diseases, idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease and shows poor prognosis. Pathological hallmark of IPF is the development of fibroblastic foci consisting of activated fibroblasts and myofibroblasts that are associated with the prognosis. The origins of such pulmonary fibroblasts are proposed to be derived from several mesenchymal cells, including originally resident pulmonary fibro....

Protocol

1. Preparations and Culture of Lung Epithelial Cells

1. Culture A549 human lung epithelial cells (adherent cell line) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin.
2. Remove and discard the cell culture media from culture dish, and wash once with 5 - 10 ml of phosphate buffered saline (PBS). After washing, immediately aspirate the PBS.
3. Add 2 ml Trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05%) and incubate at 37 °C and 5% CO₂ for 3 min.
4. Collect the detached cells in centrifuge tubes containing cell cultur....

Results

During EMT, epithelial cells lose epithelial markers, like E-cadherin, and gain the expression of mesenchymal markers, such as vimentin and α-smooth muscle actin^4,5. Incubation of A549 human lung epithelial cells with TGF-β1 and TNF-α induces EMT. The appearance of normal A549 cells are cobble stone like shape and triangle shape that is a characteristic of epithelial cells (Figure 3A), but after stimulated with TGF-β1 and TNF-α, the appearance changed to long s.......

Discussion

The protocol developed in this study comprises two steps. The first step is performed to induce EMT, while the second step is the gel contraction assay. Since it is important to confirm that cells underwent EMT, step 2 provides an excellent complement to the morphological and gene expression changes. Previous studies showed that EMT of A549 cells was induced by TGF-β1 only²⁴; however, as we have reported previously¹⁰, TNF-α treatment enhances EMT and the acquisition of mesenchymal cell mar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	sigma aldrich	11965-092	For A549 medium
FBS	GIBCO	10437
Transforming Growth Factor-β1, Human, recombinant	Wako Laboratory chemicals	209-16544
Recombinant Human TNF-α	R&D systems	210-TA/CF
E-Cadherin (24E10) Rabbit mAb	Cell Signaling Technology	#3195	1:3,000 dilution
Vimentin (D21H3) Rabbit mAb	Cell Signaling Technology	#5741	1:3,000 dilution
Anti-α-Tubulin antibody	sigma aldrich	T9026	1:10,000 dilution
Monoclonal Anti-Actin, α-Smooth Muscle antibody	sigma aldrich	A5228	1:10,000 dilution
Anti-N-cadherin antibody	BD Transduction Laboratories	#610920	1:1,000 dilution
Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)	GE Healthcare	NA931-100UL	1:20,000 dilution
Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)	GE Healthcare	NA934-100UL	1:20,000 dilution
Blocking reagent	GE Healthcare	RPN418	2% in TBS-T
6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid	corning	#353046
100 mm Cell culture dish	TPP	#93100
DMEM, powder	life technologies	12100-046	For 4× DMEM
Type 1 collagen gel	Nitta gelatin	Cellmatrix type I-A
24 Well cell culture plate	AGC TECHNO GLASS	1820-024
Gel Documentation System	ATTO	AE-6911FXN	Gel imager
Gel analyzing software	ATTO	Densitograph, ver. 3.00	analysing software bundled with AE-6911FXN
Trypsin-EDTA (0.05%), phenol red	life technologies	25300054
24 Well Plates, Non-Treated	IWAKI	1820-024
Trypan Blue Solution, 0.4%	life technologies	15250-061
RNA extraction kit	Qiagen	74106
Reverse transcriptase	life technologies	18080044
Real time PCR system	Stratagene	Mx-3000P
SYBR green PCR kit	Qiagen	204145
Protease Inhibitor Cocktail (100x)	life technologies	78429
PVDF membrane	ATTO	2392390
Protein assay kit	bio-rad	5000006JA
Polyacrylamide gel	ATTO	2331810
Western blotting detection reagent	GE Healthcare	RPN2232
Cold CCD camera	ATTO	Ez-Capture MG/ST
Trypsin inhibitor	sigma aldrich	T9003-100MG
Polyoxyethylene (20)Sorbitan Monolaurate	Wako Laboratory chemicals	163-11512
Polyoxyethylene (9) octyiphenyl ether	Wako Laboratory chemicals	141-08321