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Abstract

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Materials

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Developmental Biology

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

Published: June 10th, 2016

DOI:

10.3791/53974

1Department of Clinical Laboratory, The University of Tokyo Hospital, 2Department of Respiratory Medicine, The University of Tokyo Hospital

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

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.

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....

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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 (EDT.......

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During EMT, epithelial cells lose epithelial markers, like E-cadherin, and gain the expression of mesenchymal markers, such as vimentin and α-smooth muscle actin4,5. 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 app.......

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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.......

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We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.

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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 1:3000 dilution
Vimentin (D21H3) Rabbit mAb Cell Signaling Technology #5741 1:3000 dilution
Anti-α-Tubulin antibody sigma aldrich T9026 1:10000 dilution
Monoclonal Anti-Actin, α-Smooth Muscle antibody  sigma aldrich A5228 1:10000 dilution
Anti-N-cadherin antibody BD Transduction Laboratories #610920 1:1000 dilution
Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody) GE Healthcare NA931-100UL 1:20000 dilution
Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody) GE Healthcare NA934-100UL 1:20000 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

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