Name: Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition (Video) | JoVE Journal
Uploaded: 2016-06-10T14:00:00.000+00:00
Duration: 6 min 2 s
Description: Learn a gel contraction assay to evaluate contractile function in mesenchymal cells post-EMT using A-549 human lung epithelial cells. This accessible method requires no specialized equipment and aids inflammatory disease research.

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5 minutes

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 (EDTA) (0.05%) and incubate at 37 &#176;C and 5% CO2 for 3 min.</li>
	<li>Collect the detached cells in centrifuge tubes containing cell culture medium, centrifuge the tubes at RT for 4 min at 150 &#215; g.</li>
	<li>Resuspend the cells pellet in 2 ml of cell culture medium and remove a sample for cell counting. Count the number of the cells using a hemocytometer with Trypan blue stain to check cell viability.</li>
	<li>Seed A549 cells on 10 cm polystyrene plates at a density of 0.5 - 1.0 &#215; 106 cells/dish with 10 ml of medium for gel contraction assay. Seed the cells on 6 well polystyrene plates at a density of 0.5 - 1.0 &#215; 105 cells/well with 2 ml of medium for EMT confirmation. Incubate the cells at 37 &#176;C and 5% CO2 for 24 hr.</li>
</ol>
2. EMT Procedure
<ol>
	<li>Add 10 &#181;l of TGF-&#946;1 (5 &#181;g/ml) and 10 &#181;l of TNF-&#945; (10 &#181;g/ml) to the plate for gel contraction assay seeded in step 1.6. Add 2 &#181;l of TGF-&#946;1 (5 &#181;g/ml) and 2 &#181;l of TNF-&#945; (10 &#181;g/ml) to the plate for EMT confirmation seeded in step 1.6. Incubate at 37 &#176;C and 5% CO2 for 48 hr.</li>
</ol>
3. Confirmation of EMT Procedure by PCR and Western Blotting
<ol>
	<li>Confirm change in morphology (from cobble stone-like to spindle shape) of treated cells using phase contrast microscopy. 
	Note: Normal A549 cells have cobble stone-like and triangular shaped appearance that is a characteristic of epithelial cells, but after stimulation with TGF-&#946;1 and TNF-&#945;, the cells appear long and spindle shaped that is similar to mesenchymal cells14,15.</li>
	<li>Evaluate the expression of an epithelial marker, such as E-cadherin, and mesenchymal markers such as N-cadherin, vimentin, and &#945;-smooth muscle actin using PCR or Western blotting.
	<ol>
		<li>Extract total RNA from the cells using RNA extraction kit16 and synthesize cDNA using reverse transcriptase17 according to the manufacturer&#39;s protocol.</li>
		<li>Measure expression of mRNA levels using real time PCR system18 and monomeric cyanine dye PCR kit according to the manufacturers&#39; instructions. The specific primers for GAPDH (glyceraldehyde 3-phosphate dehydrogenase), ACTA2 (alpha-actin-2), CDH1 (cadherin-1; E-cadherin), and VIM (vimentin) are shown in Table 1.</li>
		<li>Lyse the cells using a lysis buffer (Table 2) solution containing 1% protease inhibitor cocktail. Measure all sample protein concentrations using a protein assay kit19,20, and apply the same amounts of protein to a polyacrylamide gel.
		<ol>
			<li>Perform SDS gel-electrophoresis and semi-dry transfer of the proteins to PVDF membrane21. Incubate the membrane with primary antibodies in blocking buffer for 1 - 2 hr. After incubation, wash twice the membrane with wash buffer and incubate the membrane 1 hr with second antibodies. 
			Note: See the Materials/Equipment Table for antibodies and dilutions used in these studies. See Table 2 for the components of blocking buffer.</li>
			<li>Wash the membrane with wash buffer twice. Take pictures of the membrane with the Western blotting detection kit22 using a cold CCD camera11,23.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Gel Contraction Assay for Evaluating EMT
<ol>
	<li>Aspirate the conditioned media from the cell culture vessel, and wash well with 5 - 10 ml of PBS to remove dead cells. After washing, immediately aspirate the PBS.</li>
	<li>Add 2 ml of 0.05% Trypsin/EDTA and incubate at 37 &#176;C and 5% CO2 for 3 min.</li>
	<li>Collect the detached cells in centrifuge tubes containing DMEM supplemented with trypsin inhibitor (1 mg/ml), centrifuge the tubes at RT for 4 min at 150 &#215;g.</li>
	<li>Mix type 1 collagen gel with distilled water, and 4&#215; concentrated DMEM to adjust the volumes to achieve a collagen concentration of 1.75 mg/ml, and 1&#215; DMEM concentration. Be sure to keep the gel medium on ice during this step. 
	Note: To make 6 ml of gel medium, mix well 3.5 ml of type 1 collagen gel (3 mg/ml), 1.5 ml of 4&#215; concentrated DMEM, and 1ml of distilled water.</li>
	<li>Resuspend the cell pellet in 500 &#181;l of PBS and remove a sample for cell counting. Count the number of the cells using a hemocytometer with trypan blue stain to check cell viability.</li>
	<li>Add the gel medium to adjust the volumes to achieve a cell density of 3.0 &#215; 105 cells/well (6.0 &#215; 105 cells/ml) and gently but quickly mix it by pipetting without gelation.</li>
	<li>Dispense 0.5 ml of the mixture into each well of a 24-well non-treated plate quickly and carefully to make neat cylindrical form. Be careful not to allow any air bubbles to contaminate the gels (Figure 1A). 
	Note: The gel medium containing the cells is viscous and can easily gel and form crescent shape in the well.</li>
	<li>Incubate the plate for 15 min in a cell incubator at 37 &#176;C, 5% CO2 and 95% humidity to gel completely.</li>
	<li>Detach gels from the plate without breaking by moving a sterilized spatula in a manner to draw a circumference in one direction. Using a spatula, gently transfer the gels to 60 mm tissue culture dishes containing 5 ml of DMEM/1% FBS with or without TGF-&#946;1 (5 ng/ml) and TNF-&#945; (10 ng/ml).</li>
	<li>Gently shake the dishes to ensure gels are floating on the medium. Incubate in a cell incubator at 37 &#176;C, 5% CO2 and 95% humidity.</li>
</ol>
5. Measurement of Gel Size
<ol>
	<li>Measure the collagen gel size after 0, 24, 48, and 72 hr using an image analysis system.
	<ol>
		<li>Turn on the gel documentation system (Figure 2A), and put dishes into the light shielding cabinet. Then take off the lid of dishes in the cabinet.</li>
		<li>Open the related gel analyzing software. Click the &#34;image acquisition button&#34; in the menu-bar to show the image of the gels in the cabinet. Then, click &#34;acquire&#34; in the menu bar to take pictures of the gels (Figure 2B).</li>
		<li>Click the &#34;detection button&#34; in the menu bar (Figure 2C) and adjust the measurement region (yellow circle in Figure 2C) by dragging the mouse. Then, click the &#34;auto-detect button&#34; in the menu bar (see button in Figure 2D). The software automatically detects the gels showing a heatmap (Figure 2D).</li>
		<li>Click &#34;OK&#34; to extract the outline of the gels by image processing and to calculate areas surrounded by the outline (Figure 2E). After the area is calculated, the calculated area is displayed in separate pop-up window. 
		Note: If the gels overlap each other during imaging, move gels gently using a sterile pipette tip.</li>
	</ol>
	</li>
</ol>

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</ol>

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

development of an in vitro assay to evaluate contractile function of 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 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 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 appearance changed to long s...

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 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.

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JoVE Journal

Developmental Biology

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Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

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Learn a gel contraction assay to evaluate contractile function in mesenchymal cells post-EMT using A-549 human lung epithelial cells. This accessible method requires no specialized equipment and aids inflammatory disease research.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition (Video) | JoVE Journal

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

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