Name: isolation and culture of avian embryonic valvular progenitor cells
Uploaded: 2010-10-27T17:35:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

<p class="jove_content">This research is supported by The Hartwell Foundation, the American Heart Association (Scientist Development Grant #0830384N) and the Foundation Leducq: Mitral 07CVD04.</p>

<p class="jove_title">1. Preparation</p>
<ol>
	<li>Incubate fertile quail or chicken eggs to stage 14<sup>-</sup> (about 2 days) or 25 (about 4.5 days) at 60% humidity and 37&deg;C.</li>
	<li>Prepare sterile Earl's Balanced Salt Solution (EBSS)
      <ol>
		<li>Add 100 mL 10X EBSS to 600 mL 18 M&Omega; water</li>
		<li>Add 2.2 g sodium bicarbonate</li>
		<li>Adjust the pH of the solution to 7.2</li>
		<li>Bring the solution up to 1000 mL</li>
		<li>Sterilize the solution by passing through a 0.2 &mu;m filter</li>
     </ol>
  </li>
...

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> <em style='font-style: italic'>J Morphol</em>. <b>88</b>, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> <em style='font-style: italic'>Developmental Biology</em>. <b>95</b>, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> <em style='font-style: italic'>Developmental Biology</em>. <b>302</b>, 256-256 (2007).</li></ol>

<p class="jove_content">The method for isolating endocardial cells from stage HH14<sup>-</sup> hearts originally developed by Runyan and Markwald provides a controlled, <em>in vitro</em> environment to study the factors that initiate and modulate embryonic EMT<sup>2</sup>. HH14<sup>-</sup> endocardial explants cultured without myocardium will not undergo EMT without biomechanical or biochemical intervention. If the myocardium is left on the explant it will signal a subset of endocardial cells to undergo mesenchymal transformation, but external ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
<tr>
<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
<tr>
<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
<tr>
<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
<tr>
<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
<tr>
<td>Fertile quail eggs (<em>Coturnix coturnix</em>)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (<em>Gallus gallus</em>)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

isolation and culture of avian embryonic valvular progenitor cells

<p class="jove_content">Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D <em>in vitro</em> models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.</p>

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

<p class="jove_content">This article will provide a method for isolating and culturing quail or chicken HH14<sup>-</sup> valve endocardial cells and HH25 valve cushion mesenchymal cells.</p>

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Research

JoVE Journal

Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...