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.

1. Preparations and Culture of Lung Epithelial Cells
<ol>
	<li>Culture A549 human lung epithelial cells (adherent cell line) in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 &#181;g/ml streptomycin.</li>
	<li>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.</li>
	<li>Add 2 ml&#160;Trypsin/ethylenediaminetetraacetic acid (EDT...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> The Journal of clinical investigation. 119, 1420-1428 (2009).</li><li>Forino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta1+induces+epithelial-mesenchymal+transition,+but+not+myofibroblast+transdifferentiation+of+human+kidney+tubular+epithelial+cells+in+primary+culture.">TGFbeta1 induces epithelial-mesenchymal transition, but not myofibroblast transdifferentiation of human kidney tubular epithelial cells in primary culture.</a> International journal of experimental pathology. 87, 197-208 (2006).</li><li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+miR-200+in+pulmonary+fibrosis.">Participation of miR-200 in pulmonary fibrosis.</a> The American journal of pathology. 180, 484-493 (2012).</li><li>Reynolds, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+inflammation+and+fibrosis:+an+alveolar+macrophage-centered+perspective+from+the+1970s+to+1980s.">Lung inflammation and fibrosis: an alveolar macrophage-centered perspective from the 1970s to 1980s.</a> American journal of respiratory and critical care medicine. 171, 98-102 (2005).</li><li>Camara, J., Jarai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition+in+primary+human+bronchial+epithelial+cells+is+Smad-dependent+and+enhanced+by+fibronectin+and+TNF-alpha.">Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-alpha.</a> Fibrogenesis &amp; tissue repair. 3, 2 (2010).</li><li>Kamitani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+stimulation+with+TGF-beta1+and+TNF-alpha+induces+epithelial+mesenchymal+transition+in+bronchial+epithelial+cells.">Simultaneous stimulation with TGF-beta1 and TNF-alpha induces epithelial mesenchymal transition in bronchial epithelial cells.</a> International archives of allergy and immunology. 155, 119-128 (2011).</li><li>Mikami, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphotoxin+beta+receptor+signaling+induces+IL-8+production+in+human+bronchial+epithelial+cells.">Lymphotoxin beta receptor signaling induces IL-8 production in human bronchial epithelial cells.</a> PloS one. 9, e114791 (2014).</li><li>Yamauchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha+enhances+both+epithelial-mesenchymal+transition+and+cell+contraction+induced+in+A549+human+alveolar+epithelial+cells+by+transforming+growth+factor-beta1.">Tumor necrosis factor-alpha enhances both epithelial-mesenchymal transition and cell contraction induced in A549 human alveolar epithelial cells by transforming growth factor-beta1.</a> Experimental lung research. 36, 12-24 (2010).</li><li>Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts,+myofibroblasts,+and+wound+contraction.">Fibroblasts, myofibroblasts, and wound contraction.</a> The Journal of cell biology. 124, 401-404 (1994).</li><li>Ramos, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-1+reverts+epithelial-mesenchymal+transition+induced+by+TGF-{beta}1+through+MAPK/ERK+kinase+pathway+.">FGF-1 reverts epithelial-mesenchymal transition induced by TGF-{beta}1 through MAPK/ERK kinase pathway .</a> American journal of physiology. 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The authors have no conflicts of interest to disclose.

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-&#946;1 only24; however, as we have reported previously10, TNF-&#945; treatment enhances EMT and the acquisition of mesenchymal cell mar...

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 fibroblasts and circulating fibrocytes from bone marrow. Recently, epithelial-mesenchymal transition (EMT) has been proposed to be associated with the formation of mesenchymal cells2, and to contribute to the pathogenesis of fibrotic disorders.
It is thought that EMT plays important roles in the process of fetal development, wound healing, and progression of cancer, including tumor invasion and metastasis3. Following the process of EMT, epithelial cells obtain the ability of mesenchymal cells by loss of epithelial markers, such as E-cadherin, and by expression of mesenchymal markers, such as vimentin, and &#945;-smooth muscle actin (SMA)4,5. Previous studies showed the evidence that EMT process has been associated with the development of tissue fibrosis in the kidney6 and lung7. Additionally, chronic inflammation promotes fibrotic disease8; furthermore, such inflammatory cytokines as Tumor necrosis factor superfamily member 14 (TNFSF14; LIGHT), tumor necrosis factor (TNF)-&#945;, and interleukin-1&#946;, have been shown to enhance EMT9-12.
Collagen gel contraction assay, a collagen-based cell contraction assay in which fibroblasts are embedded in type I collagen gel three-dimensionally, is an ideal in vitro model for the evaluation of contractility. Contractility is one of the characteristic functions of fibroblasts and contributes to normal wound repair and fibrosis13. In this assay, it is thought that the attachment of fibroblasts to type I collagen through integrin-dependent mechanisms is supposed to produce mechanical tension under some conditions, and consequently lead to tissue contraction.
Here, the development of the gel contraction assay is reported to be adapted to evaluate the acquisition of contractile function in the cells that underwent EMT. This report demonstrates that this modified assay is suitable for evaluating contractility in mesenchymal cells that underwent EMT.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:167px;">For A549 medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:96px;">GIBCO</td>
			<td style="width:119px;">10437</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Transforming Growth Factor-&#946;1, Human, recombinant</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">209-16544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human TNF-&#945;</td>
			<td style="width:96px;">R&#38;D systems</td>
			<td style="width:119px;">210-TA/CF</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-Cadherin (24E10) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#3195</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vimentin (D21H3) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#5741</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-&#945;-Tubulin antibody</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9026</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle antibody&#160;</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">A5228</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-N-cadherin antibody</td>
			<td style="width:96px;">BD Transduction Laboratories</td>
			<td style="width:119px;">#610920</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA931-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">blocking reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN418</td>
			<td>2% in TBS-T</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid</td>
			<td style="width:96px;">corning</td>
			<td style="width:119px;">#353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mm cell culture dish</td>
			<td style="width:96px;">TPP</td>
			<td style="width:119px;">#93100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, powder</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">12100-046</td>
			<td>For 4&#215;DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">type 1 collagen gel</td>
			<td style="width:96px;">Nitta gelatin</td>
			<td style="width:119px;">Cellmatrix type I-A</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">24 well cell culture plate</td>
			<td style="width:96px;">AGC TECHNO GLASS</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Documentation System&#160;</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">AE-6911FXN</td>
			<td>Gel imager</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">gel analyzing software</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">Densitograph, ver. 3.00</td>
			<td>analysing software bundled with AE-6911FXN</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 Well Plates, Non-Treated</td>
			<td style="width:96px;">IWAKI</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNA extraction kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reverse transcriptase</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">18080044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">real time PCR system</td>
			<td style="width:96px;">Stratagene</td>
			<td style="width:119px;">Mx-3000P</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR green PCR kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">204145</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Protease Inhibitor Cocktail (100X)</td>
			<td style="width:96px;">life technologies</td>
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			<td style="width:119px;">2392390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">protein assay kit</td>
			<td style="width:96px;">bio-rad</td>
			<td style="width:119px;">5000006JA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylamide gel</td>
			<td style="width:96px;">ATTO</td>
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			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">163-11512</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">polyoxyethylene (9) octyiphenyl ether</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">141-08321</td>
			<td></td>
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</table>

development of an in vitro assay to evaluate contractile function of 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 &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. 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.

During EMT, epithelial cells lose epithelial markers, like E-cadherin, and gain the expression of mesenchymal markers, such as vimentin and &#945;-smooth muscle actin4,5. Incubation of A549 human lung epithelial cells with TGF-&#946;1 and TNF-&#945; 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-&#946;1 and TNF-&#945;, the app...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

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

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

Development of an In Vitro Assay to Evaluate Contractile Function of 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 overall goal of this procedure is to evaluate the contractility of mesenchymal cells that have undergone in vitro inflamatory cytokine induced epithelial mesenchymal transition, or EMT. This mesenchymal is a variation of basic cell function after EMT induced by inflammatory immune responses such as during idiopathic pulmonary fibrosis or, airway remodeling in severe asthma. The main advantages of this technique are that it does not require specific devices and that it demonstrates a high content Demontrating the procedure will be Hideyuki Takeshima and Kosuke Makita graduate students from my laboratory.Begin by washing a culture of A-549 human lung epithelial cells with five to ten millileters of PBS. Next, detach the adherent cells with two millileters of trypsin-EDTA at 37 degrees Celsius and five percent carbon dioxide. After three minutes, transfer the cell suspension into conical tubes containing cell culture medium and spin down the cells in a centrifuge.Resuspend the pellets in two millileters of cell culture medium, reserving a small eloquate for counting. Then seed 0.5 to 1 times 10 to the 6th cells in ten millileters of medium per ten centimeter polysterene dish and incubate the cell cultures for 24 hours. For EMT induction, the next day, treat the cells with 10 microliters of TGF beta 1 and TNF alpha, and return the plates to the incubator for another 48 hours.On day four, wash the emt induced cultures with five to ten millileters of pbs, then detach cells with trypsin-EDTA as just demonstrated. Next, spin down the cell suspensions in DMEM supplemented with tryspsin inhibitor and resuspend the pellets in 500 microliters of pbs. Then count the cells, and add freshley prepared gell medium to a density of 3 times 10 to the 5th cells per well, gently but quickly mixing the solution by pipette before gelation.Promptly, but carefully, dispense 0.5 millileters of cells into each well of a non-treated 24 well plate in neat, cylindrical forms. Then place the plate in a cell culture incubator for 15 minutes. When the gel has completely solidified, move a sterilized spatuala in a circle around the outside of each gel in one direction, to detach the gels from the plates without breaking.When removing the gel, be careful not to move the spatula back and forth in the well. Then use the spatula to gently transfer the gels to individual 16 millimeter tissue culture dishes containing five millileters of DMEM supplemented with one percentFBS, with or without TGF beta 1 and TNF alpha. Finally, gently shake the dishes to ensure that the gels are floating on the medium, and return the gels to the cell culture incubator.Untreated a541 cells display a cobblestone like and triangle shaped appearance, characteristic of epithelial cells. After their stimulation with TGF beta 1 and TNF alpha however, the cells demonstrate a long spindle shape, similar to that observed for mesynchemal cells. QRTCPR of the epithelial and mesynchemal marker expression before and after EMT reveals that A541 cells treated with inflammatory cytokines for 48 hours exhibit a significantly reduced expression of CDH1 and significantly increased expressions of VIAM and ACTA2.Further, stimulation with these cytokines attenuates E-cadherin expression as determined by western blood analysis, whereas the expressions of vimentin N-cadherin, and alpha smooth muscle actin are induced. In a gel contraction assay, after 48 hours, the gels containing TGF beta 1 and TNF alpha treated cells are smaller than the controlled gels containing cells treated with PBS. Indeed, after 72 hours, the size of the gels containing the cytokine treated gels is significantly reduced, compared to the controlled gels as measured by a gel analyzer.As observed, the gel contraction steps can be completed in three hours if they are performed properly. While attempting this procedure, it's important to remember to confirm that the EMT has been successfully induced before performing the gel contracting assay. After observement, this technique provides a way for a variating the EMT process during inflammatory state such as fibrosis or lung remodeling.After watching this video, you should have a good understanding of how to emit cells that have EMT, into a type of cuagenter and to froth them in medium.

development-an-vitro-assay-to-evaluate-contractile-function

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JoVE Journal

Developmental Biology

12.0K Görüntüleme.  The University of Tokyo Hospital. The overall goal of this procedure is to evaluate the contractility of mesenchymal cells that have undergone in vitro inflamatory cytokine induced epithelial mesenchymal transition, or EMT. This mesenchymal is a variation of basic cell function after EMT induced by inflammatory immune responses such as during idiopathic pulmonary fibrosis or, airway remodeling in severe asthma. The main advantages of this technique are that it does not require specific devices and that it demonstrates a high ...