This work was supported by funds from DUKE-NUS Graduate Medical School Singapore, Goh foundation and Singapore NRF fellowship (NRF-NRFF2016-01) to Manvendra K. Singh.

All experiments were approved by the Duke-NUS Graduate Medical School Institutional Animal Care and Use Committee.
1. Retrieving the Embryonic Ventricles
<ol>
	<li>Sacrifice a timed pregnant mouse at the desired embryonic stage (E11.5 or E12.5) using a euthanasia chamber with carbon dioxide gas supply or another approved euthanasia method.</li>
	<li>Place the mouse on its back on a dissecting table. Disinfect the abdomen of the female with 70% ethanol. Lift the skin over the belly and make a small incision (1-2 cm) with a blade, then hold the skin with both hands and pull away to expose the abdominal wall.</li>
	<li>Now cut the abdominal wall to expose the uterine horn. Use sterile forceps to lift up the uterine horn and carefully cut away the fat tissues and blood vessels attached to the uterine horn. Cut at the cervix to retrieve the uterus.</li>
	<li>Place the uterine horn in a petri dish containing sterile cold 1x Phosphate Buffered Saline (PBS) and rinse gently. Place the petri dish on ice.</li>
	<li>Use scissors to cut through the midline of the uterine horn (opposite to the site where the placenta is located) to expose the embryos that are still inside the yolk sac and attached to the uterine wall through the placenta.</li>
	<li>Using sterile forceps, cut open the embryonic yolk sac to free the embryo.</li>
	<li>Place the embryo in a new petri dish containing sterile cold 1x PBS. Decapitate the embryo and place it on its back. Cut open the chest wall to expose the heart.</li>
	<li>Use sterile forceps to lift the heart and cut away the vessels around it to free the heart from the chest wall. Pay special attention not to damage the epicardium of the heart with the surgical tools.</li>
	<li>Trim off the outflow tract and both atria. Transfer the ventricle to another dish with cold 1x PBS. Place the dish on ice.</li>
	<li>Repeat steps 1.6-1.9 till all the ventricles are retrieved.</li>
</ol>
2. Epicardial Explant Culture
Note: Culture these epicardial explants either on glass chamber slides or on collagen gel for downstream applications.
<ol>
	<li>Glass Chamber Slides
	<ol>
		<li>To prepare the culture media, add 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 ng/ml recombinant fibroblast growth factor 2 (FGF2) to Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). Perform all the steps in the laminar flow hood.</li>
		<li>Add 500 &#181;l culture media to each well of an 8-well chamber.</li>
		<li>Place each excised ventricle in the center of a well. Orient the ventricle to keep the dissected side down on the bottom surface of the well.</li>
		<li>Gently put the plate in a 37 &#176;C, 5% CO2 cell culture incubator.</li>
		<li>Maintain the culture with minimum disturbance to allow the explants to adhere. Formation of the epicardial monolayer can be observed after 48-72 hr.</li>
		<li>For differentiation of epicardial cells into smooth muscle cells, culture the epicardial monolayer cells in differentiation media for another 6 days. To prepare the differentiation media, add 10% FBS, 1% penicillin/streptomycin and 50 ng/ml recombinant transforming growth factor beta 1 (TGF-&#946;1) to DMEM.</li>
		<li>Change the culture media on alternate days.</li>
	</ol>
	</li>
	<li>Collagen Gel
	<ol>
		<li>Prepare the collagen gel in a 96-well plate using a commercial 3D Collagen Culture kit according to manufacturer&#39;s protocol.</li>
		<li>To prepare the desired volume of collagen gel, mix the appropriate amount of collagen solution with 5x of DMEM and pipette up and down. The solution should turn yellow.</li>
		<li>Add a corresponding volume of neutralization solution and mix well immediately by pipetting up and down. The solution will turn pink.</li>
		<li>Pipette 100 &#181;l of this mixture into each well of a 96-well plate. Place the plate in a 37 &#176;C, 5% CO2 incubator for 30-60 min to allow the gel to polymerize.</li>
		<li>Add 200 &#181;l of culture media described above to each well.</li>
		<li>Place each excised ventricle in the center of a well. Orient the ventricle to keep the dissected side down on the collagen gel (apex of the ventricle should face the experimenter). Gently put the plate back in the cell culture incubator.</li>
		<li>After 3 days, remove the ventricles. Put the plate back into the cell culture incubator for another 2 days.</li>
		<li>Change the culture media on alternate days.</li>
	</ol>
	</li>
</ol>
3. Fixation and Visualization
Note: Epicardial cells can be visualized on either glass chamber slides or collagen gel.
<ol>
	<li>Aspirate out the culture media and add an equal volume of sterile 1x PBS to wash the cells.</li>
	<li>Add just enough 4% paraformaldehyde (PFA) to cover the cells. Fix the cells for 10 min at 4 &#176;C.</li>
	<li>Remove PFA and wash explants with 1x PBST (1x PBS with 0.1% triton X-100) to permeabilize the cells.</li>
	<li>Immunostain with desired antibody (e.g. Alexa Fluor 488-phalloidin; ZO-1, &#945;-Tubulin and &#945;-smooth muscle actin)7.</li>
</ol>

<ol>
	<li>Mikawa, T., Fischman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+analysis+of+cardiac+morphogenesis:+discontinuous+formation+of+coronary+vessels.">Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels.</a> Proc Natl Acad Sci U S A. 89, 9504-9508 (1992).</li><li>Mikawa, T., Gourdie, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericardial+mesoderm+generates+a+population+of+coronary+smooth+muscle+cells+migrating+into+the+heart+along+with+ingrowth+of+the+epicardial+organ.">Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ.</a> Dev Biol. 174, 221-232 (1996).</li><li>Manner, J., Perez-Pomares, J. M., Macias, D., Munoz-Chapuli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin,+formation+and+developmental+significance+of+the+epicardium:+a+review.">The origin, formation and developmental significance of the epicardium: a review.</a> Cells Tissues Organs. 169, 89-103 (2001).</li><li>Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Mentink, M. M., Gourdie, R. G., Poelmann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium-derived+cells+contribute+a+novel+population+to+the+myocardial+wall+and+the+atrioventricular+cushions.">Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions.</a> Circ Res. 82, 1043-1052 (1998).</li><li>von Gise, A., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+and+epicardial+epithelial+to+mesenchymal+transitions+in+heart+development+and+disease.">Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease.</a> Circ Res. 110, 1628-1645 (2012).</li><li>Katz, T. 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=Distinct+compartments+of+the+proepicardial+organ+give+rise+to+coronary+vascular+endothelial+cells.">Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells.</a> Dev Cell. 22, 639-650 (2012).</li><li>Singh, M. K., Lu, M. M., Massera, D., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-processing+enzyme+Dicer+is+required+in+epicardium+for+coronary+vasculature+development.">MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development.</a> J Biol Chem. 286, 41036-41045 (2011).</li><li>Degenhardt, K., Singh, M. K., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+under+development:+cardiovascular+embryology+applied+to+heart+disease.">New approaches under development: cardiovascular embryology applied to heart disease.</a> J Clin Invest. 123, 71-74 (2013).</li><li>Singh, M. K., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium-derived+cardiac+mesenchymal+stem+cells:+expanding+the+outer+limit+of+heart+repair.">Epicardium-derived cardiac mesenchymal stem cells: expanding the outer limit of heart repair.</a> Circ Res. 110, 904-906 (2012).</li><li>Manner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+on+the+formation+of+the+epicardium+in+chick+embryos.">Experimental study on the formation of the epicardium in chick embryos.</a> Anat Embryol (Berl). 187, 281-289 (1993).</li><li>Manner, J., Schlueter, J., Brand, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+analyses+of+the+function+of+the+proepicardium+using+a+new+microsurgical+procedure+to+induce+loss-of-proepicardial-function+in+chick+embryos.">Experimental analyses of the function of the proepicardium using a new microsurgical procedure to induce loss-of-proepicardial-function in chick embryos.</a> Dev Dyn. 233, 1454-1463 (2005).</li><li>Pennisi, D. J., Ballard, V. L., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium+is+required+for+the+full+rate+of+myocyte+proliferation+and+levels+of+expression+of+myocyte+mitogenic+factors+FGF2+and+its+receptor,+FGFR-1,+but+not+for+transmural+myocardial+patterning+in+the+embryonic+chick+heart.">Epicardium is required for the full rate of myocyte proliferation and levels of expression of myocyte mitogenic factors FGF2 and its receptor, FGFR-1, but not for transmural myocardial patterning in the embryonic chick heart.</a> Dev Dyn. 228, 161-172 (2003).</li><li>Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Bergwerff, M., Mentink, M. M., Poelmann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+outgrowth+inhibition+leads+to+compensatory+mesothelial+outflow+tract+collar+and+abnormal+cardiac+septation+and+coronary+formation.">Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation.</a> Circ Res. 87, 969-971 (2000).</li><li>Lavine, K. J., 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=Endocardial+and+epicardial+derived+FGF+signals+regulate+myocardial+proliferation+and+differentiation+in+vivo.">Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo.</a> Dev Cell. 8, 85-95 (2005).</li><li>Merki, E., 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=Epicardial+retinoid+X+receptor+alpha+is+required+for+myocardial+growth+and+coronary+artery+formation.">Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation.</a> Proc Natl Acad Sci U S A. 102, 18455-18460 (2005).</li><li>Stuckmann, I., Evans, S., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+and+retinoic+acid,+secreted+from+the+epicardium,+are+required+for+cardiac+myocyte+proliferation.">Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation.</a> Dev Biol. 255, 334-349 (2003).</li><li>Huang, G. N., 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=C/EBP+transcription+factors+mediate+epicardial+activation+during+heart+development+and+injury.">C/EBP transcription factors mediate epicardial activation during heart development and injury.</a> Science. 338, 1599-1603 (2012).</li><li>Zhou, B., 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=Adult+mouse+epicardium+modulates+myocardial+injury+by+secreting+paracrine+factors.">Adult mouse epicardium modulates myocardial injury by secreting paracrine factors.</a> J Clin Invest. 121, 1894-1904 (2011).</li><li>Christoffels, V. 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=Tbx18+and+the+fate+of+epicardial+progenitors.">Tbx18 and the fate of epicardial progenitors.</a> Nature. 458, 8-9 (2009).</li><li>Rudat, C., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wt1+and+epicardial+fate+mapping.">Wt1 and epicardial fate mapping.</a> Circ Res. 111, 165-169 (2012).</li><li>Duim, S. N., Kurakula, K., Goumans, M. J., Kruithof, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+endothelial+cells+express+Wilms'+tumor-1:+Wt1+expression+in+the+developing,+adult+and+infarcted+heart.">Cardiac endothelial cells express Wilms' tumor-1: Wt1 expression in the developing, adult and infarcted heart.</a> J Mol Cell Cardiol. 81, 127-135 (2015).</li><li>Iyer, D., 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=Robust+derivation+of+epicardium+and+its+differentiated+smooth+muscle+cell+progeny+from+human+pluripotent+stem+cells.">Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells.</a> Development. 142, 1528-1541 (2015).</li><li>Witty, A. D., 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=Generation+of+the+epicardial+lineage+from+human+pluripotent+stem+cells.">Generation of the epicardial lineage from human pluripotent stem cells.</a> Nat Biotechnol. 32, 1026-1035 (2014).</li></ol>

The authors declare that they have no competing financial interests.

It is pivotal to develop techniques that facilitate the study of the epicardium to cater to the increasing significance of the epicardium in cardiac development and regeneration. The epicardial culture system poses significant advantages for epicardial research.
An alternative way to isolate epicardial cells is to use fluorescence-activated cell sorting (FACS). This method relies on the use of epicardial markers (or epicardium-cell-specific transgenic expression of a fluorescent protein) to se...

A wealth of experimental data have illustrated that the epicardium influences critical steps in cardiac development. During development, the septum transversum gives rise to a clump of mesothelial cells known as the proepicardium1-4. Cells from the proepicardium then migrate and envelope the myocardium forming the epicardium. Following this, a subset of epicardial cells undergo EMT giving rise to a migratory population of epicardium-derived cells (EPDCs) that subsequently invade the myocardium. Genetic as well as retroviral lineage tracing experiments have demonstrated that EPDCs differentiate into various lineages including smooth muscle cells, fibroblasts, endothelial cells and cardiomyocytes (if any). Therefore EPDCs contribute significantly to the development of the coronary vasculature and myocardial architecture1,2,4-9. Furthermore, the epicardium is essential for the development of the ventricular compact layer10-12. For example Gittenberger-de Groot et al. demonstrated that inhibiting the outgrowth of the proepicardium leads to an array of defects such as a thin myocardium, deficient looping of the heart and abnormal interventricular septum formation and as a result, embryonic lethality13. Paracrine factors secreted from the embryonic epicardium modulate cardiomyocyte proliferation and differentiation. Consistent with this, epicardium-specific deletion of signaling pathways such as retinoic acid (RA), fibroblast growth factors (FGFs) and Wnt/&#946;-catenin resulted in defective myocardial growth and embryonic lethality14-16.
Though the epicardium was believed to be quiescent in adult hearts, recent studies have shown that the developmental program is reactivated in the epicardium following cardiac injury17,18. Upon activation, the cells undergo rapid proliferation and EMT that result in EPDC formation. These cells exhibit the capacity to differentiate into fibroblast and smooth muscle cells but not cardiomyocytes or endothelial cells18. In addition, the EPDCs secrete proangiogenic factors that aid in the vascularization of the injured area and thus facilitate improved cardiac function by reducing the infarct size. Due to these findings, the epicardium has gained interest in the study of cardiovascular development, disease and regeneration.
Transgenic technology has revolutionized medical research in the 21st century. With the aid of transgenic technologies, diseased mouse models mimicking the human condition metabolically and pathophysiologically have been successfully developed. However, studying the epicardial cell behavior in these mutants has been a challenge mainly due to early embryonic lethality. Considering the significant role that the epicardium plays in cardiac development and regeneration, we have established an in vitro culture system for mouse epicardial cells. This method allows the long term culture of the epicardial cells and facilitates the detailed study of the two important properties of the epicardium: its ability to migrate and differentiate. The excised ventricles from the mouse could be cultured on collagen gels which can be used to conduct migration assays. Being cultured in a 3D matrix that replicates the collagen-rich extracellular matrix of the subepicardial layer better recapitulates the in vivo cell physiology. Alternatively they can be cultured on chamber slides in order to establish an epicardial monolayer which can then be used for a variety of downstream applications. This monolayer can be used to stain for tight junction proteins which will provide insights on the ability of the epicardium to undergo EMT which is crucial for migration. In addition, differentiation experiments can also be carried out on these cells. Furthermore, gene expression profile can be analyzed by extracting RNA from the cells and performing quantitative polymerase chain reaction (qPCR). Lastly, the monolayers could also be treated with agents followed by a molecular analysis to test for potential therapeutics. Put together, this epicardial culture system provides us with the opportunity to visualize and gather molecular data which furthers our understanding about epicardial development.
Another desirable feature of this method is that it is straightforward and no elaborate setup is required. In brief, the embryos are harvested at E11.5 or E12.5 following which the heart is excised. The ventricles are then cultured on either collagen gel or chamber slides. Subsequently, these cells can be used to conduct downstream experiments.

<table border="0" cellpadding="0" cellspacing="0" width="708">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:131px;">Life tech invitrogen&#160;</td>
			<td style="width:131px;">11995065</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin solution</td>
			<td>Life tech invitrogen&#160;</td>
			<td>15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)&#160;</td>
			<td>Life tech invitrogen&#160;</td>
			<td>10500064</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148-5KG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant fibroblast growth factor 2 (FGF2)</td>
			<td style="width:131px;">PeproTech</td>
			<td style="width:131px;">450-33</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant transforming growth factor beta 1 (TGF-&#946;1)</td>
			<td style="width:131px;">PeproTech</td>
			<td style="width:131px;">100-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">ZO-1 antibody</td>
			<td>Life tech invitrogen&#160;</td>
			<td style="width:131px;">40-2200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Tubulin antibody</td>
			<td>Sigma</td>
			<td style="width:131px;">T 6074</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">&#945;-smooth muscle actin (SMA) antibody</td>
			<td>Sigma</td>
			<td style="width:131px;">A 2547</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Phalloidin antibody</td>
			<td>Life tech invitrogen&#160;</td>
			<td style="width:131px;">A12379</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D Collagen Culture kit&#160;</td>
			<td>Millipore&#160;</td>
			<td>ECM 675</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-well chamber slide</td>
			<td>Fisher Scientific</td>
			<td>NNU 154534-PK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Trizol reagent</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:131px;">15596-018</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:131px;">Life Technologies</td>
			<td>4453536</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superscript First Strand Synthesis kit</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:131px;">11904-018</td>
		</tr>
	</tbody>
</table>

in vitro culture of epicardial cells from mouse embryonic heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

Using this culture protocol, primary epicardial cells can be isolated with high purity for downstream applications. The cultured cells are able to undergo EMT, migrate and differentiate just as epicardial cells do in vivo.
To determine the purity of our primary epicardial cell culture, we analyzed the epicardial explants generated from Sema3dGFPCre/+;R26Tom/+ emb...

Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

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7.3K Views.  Duke-NUS Graduate Medical School. The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. 

Video: In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen 1-7. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion2. Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation8-12. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition8, 10, 13, 14 ("coking") and sulfur poisoning11, 15 and the manner in which surface modifications stave off this degradation16. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated17-22. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H2S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.

We present a unique platform for characterizing electrode surfaces in solid oxide fuel cells (SOFCs) that allows simultaneous performance of multiple characterization techniques (e.g. in situ Raman spectroscopy and scanning probe microscopy alongside electrochemical measurements). Complementary information from these analyses may help to advance toward a more profound understanding of electrode reaction and degradation mechanisms, providing insights into rational design of better materials for SOFCs.

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Integration of Light Trapping Silver Nanostructures in Hydrogenated Microcrystalline Silicon Solar Cells by Transfer Printing

One of the potential applications of metal nanostructures is light trapping in solar cells, where unique optical properties of nanosized metals, commonly known as plasmonic effects, play an important role. Research in this field has, however, been impeded owing to the difficulty of fabricating devices containing the desired functional metal nanostructures. In order to provide a viable strategy to this issue, we herein show a transfer printing-based approach that allows the quick and low-cost integration of designed metal nanostructures with a variety of device architectures, including solar cells. Nanopillar poly(dimethylsiloxane) (PDMS) stamps were fabricated from a commercially available nanohole plastic film as a master mold. On this nanopatterned PDMS stamps, Ag films were deposited, which were then transfer-printed onto block copolymer (binding layer)-coated hydrogenated microcrystalline Si (&#181;c-Si:H) surface to afford ordered Ag nanodisk structures. It was confirmed that the resulting Ag nanodisk-incorporated &#181;c-Si:H solar cells show higher performances compared to a cell without the transfer-printed Ag nanodisks, thanks to plasmonic light trapping effect derived from the Ag nanodisks. Because of the simplicity and versatility, further device application would also be feasible thorough this approach.

A viable transfer printing-based methodology to introduce plasmonic metal nanostructures in solar cells is described. Using nanopillar poly(dimethylsiloxane) stamps, an Ag-based ordered nanodisk array was integrated with standard hydrogenated microcrystalline Si solar cells, which led to improved device performances due to plasmonic light trapping.

One of the potential applications of metal nanostructures is light trapping in solar cells, where unique optical properties of nanosized metals, commonly ...

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization

Polymer-based materials hold promise as low-cost, flexible efficient photovoltaic devices. Most laboratory efforts to achieve high performance devices have used devices prepared by spin coating, a process that is not amenable to large-scale fabrication. This mismatch in device fabrication makes it difficult to translate quantitative results obtained in the laboratory to the commercial level, making optimization difficult. Using a mini-slot die coater, this mismatch can be resolved by translating the commercial process to the laboratory and characterizing the structure formation in the active layer of the device in real time and in situ as films are coated onto a substrate. The evolution of the morphology was characterized under different conditions, allowing us to propose a mechanism by which the structures form and grow. This mini-slot die coater offers a simple, convenient, material efficient route by which the morphology in the active layer can be optimized under industrially relevant conditions. The goal of this protocol is to show experimental details of how a solar cell device is fabricated using a mini-slot die coater and technical details of running in situ structure characterization using the mini-slot die coater.

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

Polymer-based materials hold promise as low-cost, flexible efficient photovoltaic devices. Most laboratory efforts to achieve high performance devices ...

Fabrication of White Light-emitting Electrochemical Cells with Stable Emission from Exciplexes

The authors present an approach for fabricating stable white light emission from polymer light-emitting electrochemical cells (PLECs) having an active layer which consists of blue-fluorescent poly(9,9-di-n-dodecylfluorenyl-2,7-diyl) (PFD) and &#960;-conjugated triphenylamine molecules. This white light emission originates from exciplexes formed between PFD and amines in electronically excited states. A device containing PFD, 4,4&#39;,4&#39;&#39;-tris[2-naphthyl(phenyl)amino]triphenylamine (2-TNATA), Poly(ethylene oxide) and K2CF3SO3 showed white light emission with Commission internationale de l&#39;&#233;clairage (CIE) coordinates of (0.33, 0.43) and a Color Rendering Index (CRI) of Ra = 73 at an applied voltage of 3.5 V. Constant voltage measurements showed that the CIE coordinates of (0.27, 0.37), Ra of 67, and the emission color observed immediately after application of a voltage of 5 V were nearly unchanged and stable after 300 sec.

The authors present a method for fabricating stable white-light-emitting electrochemical cells utilizing emission from exciplexes formed between a blue-emitting fluorene polymer and aromatic amines.

The authors present an approach for fabricating stable white light emission from polymer light-emitting electrochemical cells (PLECs) having an active ...

In Situ Monitoring of the Accelerated Performance Degradation of Solar Cells and Modules: A Case Study for Cu(In,Ga)Se2 Solar Cells

The levelized cost of electricity (LCOE) of photovoltaic (PV) systems is determined by, among other factors, the PV module reliability. Better prediction of degradation mechanisms and prevention of module field failure can consequently decrease investment risks as well as increase the electricity yield. An improved knowledge level can for these reasons significantly decrease the total costs of PV electricity. 
In order to better understand and minimize the degradation of PV modules, the occurring degradation mechanisms and conditions should be identified. This should preferably happen under combined stresses, since modules in the field are also simultaneously exposed to multiple stress factors. Therefore, two 'Combined Stress test with in situ measurement' setups have been designed and constructed. These setups allow the simultaneous use of humidity, temperature, illumination, and electrical biases as independently controlled stress factors on solar cells and minimodules. The setups also allow real-time monitoring of the electrical properties of these samples. This protocol presents these setups and describes the experimental possibilities. Moreover, results obtained with these setups are also presented: various examples about the influence of both deposition and degradation conditions on the stability of thin film Cu(In,Ga)Se2 (CIGS) as well as Cu2ZnSnSe4 (CZTS) solar cells are described. Results on the temperature dependency of CIGS solar cells are also presented.

In Situ Monitoring of the Accelerated Performance Degradation of Solar Cells and Modules: A Case Study for CuIn,GaSe2 Solar Cells

Two &#39;Combined Stress test with in situ measurement&#39; setups, which allow real-time monitoring of accelerated degradation of solar cells and modules, were designed and constructed. These setups allow the simultaneous use of humidity, temperature, electrical biases, and illumination as independently controlled stress factors. The setups and various experiments executed are presented.

The levelized cost of electricity (LCOE) of photovoltaic (PV) systems is determined by, among other factors, the PV module reliability. Better prediction ...

Fabrication of Ultra-thin Color Films with Highly Absorbing Media Using Oblique Angle Deposition

Ultra-thin film structures have been studied extensively for use as optical coatings, but performance and fabrication challenges remain.&#160; We present an advanced method for fabricating ultra-thin color films with improved characteristics. The proposed process addresses several fabrication issues, including large area processing. Specifically, the protocol describes a process for fabricating ultra-thin color films using an electron beam evaporator for oblique angle deposition of germanium (Ge) and gold (Au) on silicon (Si) substrates.&#160; Film porosity produced by the oblique angle deposition induces color changes in the ultra-thin film. The degree of color change depends on factors such as deposition angle and film thickness. Fabricated samples of the ultra-thin color films showed improved color tunability and color purity. In addition, the measured reflectance of the fabricated samples was converted into chromatic values and analyzed in terms of color. Our ultra-thin film fabricating method is expected to be used for various ultra-thin film applications such as flexible color electrodes, thin film solar cells, and optical filters. Also, the process developed here for analyzing the color of the fabricated samples is broadly useful for studying various color structures.

We present a detailed method for fabricating ultra-thin color films with improved characteristics for optical coatings. The oblique angle deposition technique using an electron beam evaporator allows improved color tunability and purity. Fabricated films of Ge and Au on Si substrates were analyzed by reflectance measurements and color information conversion.

Ultra-thin film structures have been studied extensively for use as optical coatings, but performance and fabrication challenges remain.&#160; We present ...

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely M&#246;ssbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. M&#246;ssbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

Here, we present a protocol to describe ex situ and in situ investigations of structural transformations in metallic glasses. We employed nuclear-based analytical methods which inspect hyperfine interactions. We demonstrate the applicability of M&#246;ssbauer spectrometry and nuclear forward scattering of synchrotron radiation during temperature-driven experiments.

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic ...

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, wound healing, root canal treatment, etc., forming a new research field named plasma medicine. Being a mixture of electrical, chemical, and biological reactive species, CAPs have shown their abilities to enhance nerve stem cells differentiation both in vitro and in vivo and are becoming a promising way for neurological disease treatment in the future. The much more exciting news is that using CAPs may realize one-step, and safely directed, differentiation of neural stem cells (NSCs) for tissue transportation. We demonstrate here the detailed experimental protocol of using a self-made CAP jet device to enhance NSC differentiation in C17.2-NSCs and primary rat neural stem cells, as well as observing the cell fate by inverted and fluorescence microscopy. With the help of immunofluorescence staining technology, we found both the NSCs showed an accelerated differential rate than the untreated group, and ~75% of the NSCs selectively differentiated into neurons, which are mainly mature, cholinergic, and motor neurons.

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, ...