Name: isolation of embryonic ventricular endothelial cells
Uploaded: 2013-07-20T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Embryonic Ventricular Endothelial Cells at JoVE.com

We would like to thank Andrea Portbury for critical reading of the manuscript, the Microscope Service Laboratory of UNC for assistance with the time-lapse imaging, and the NIH (grant # R01HL061656) for funding support.

1. Prepare Antibody-conjugated Beads
<ol>
 <li>One day prior to dissection, combine the antibody of choice with the appropriate Dynabeads (e.g. rat IgG Dynabeads for an antibody raised in rat, 4 x 108 beads/ml) per the manufacturer's instructions. For the BD rat anti-CD31 antibody (0.5 &mu;g/&mu;l, used to isolate endothelial cells), 3 &mu;l antibody is added for every 25 &mu;l beads, for a final concentration of 1.5 &mu;g anti-CD31 antibody and 1 x 107 beads/25 &mu;l buffer.</li>
 <li>In...

<ol>
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Working with primary embryonic cells allows novel experiments to address in vitro critical steps of development. However, the isolation procedure is not trivial. Critical steps for isolating any type of cells include ensuring that the cells are well separated upon collagenase digestion and then that they are well suspended during the wash steps. Mechanical dissection by pipetting greatly helps separate the cells during the collagenase step, and the filtering step removes clumps of cells. These steps impro...

The advent of immortalized cell lines has revolutionized basic cell biology 1. The currently available cell lines are derived from a wide range of organs and encompass all the major cell types. However, established cell lines have some limitations. The process of immortalization obviously changes the behavior of the cells, specifically with respect to life span and proliferation, but can also affect the expression of unexpected proteins, such as cytoskeletal proteins 2. In addition, although many different cell lines are available, there is significant diversity among even a single cell type within an entire organism. Endothelial cells in particular show diverse behaviors based on whether they line arteries or veins and what kind of flow is present in the vessel 3. Even more pressing from a developmental perspective, however, is that the vast majority of the available cell lines are derived from adult tissue (see, e.g. the collection available via ATCC). These adult cell lines likely do not recapitulate the dynamic nature of their embryonic precursors. The rapidly changing spatiotemporal gene expression patterns observed during development also suggest that an endothelial cell from one organ at a given developmental stage may not behave the same way as an endothelial cell from that same organ at a different developmental stage.
As an alternative to using commercially available immortalized cell lines, primary cells can be isolated from the specific tissue of interest. Among the advantages of this technique, these primary cells can be isolated from a specific organ or even part of an organ at any specific developmental stage. Further, these primary cells can be isolated from the wide variety of available transgenic animals, allowing the in vitro study of gene knockout and knock-in while avoiding other problems such as transfection efficiency. Not surprisingly, many techniques have been published detailing how to isolate specific cell types 4,5. In general, these techniques involve collecting the region of interest, dissociating the cells, tagging the specific cell type of interest, and isolating those cells for further analysis.
To study an early embryonic population of coronary endothelial cells, we scaled down a previously published technique4 for use with a smaller organ. With this scaled-down procedure, we can isolate the coronary endothelial cells from the embryonic heart at specific embryonic stages. These cells can then be used in traditional endothelial assays, such as migration analyses. Until early embryonic cell lines become more prevalent, working with the primary cells is an invaluable technique.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Timed-pregnant mice</td>
			<td></td>
			<td></td>
			<td>To be dissected at the embryonic stage of interest</td>
		 </tr>
		 <tr>
			<td>Anti-mouse CD31</td>
			<td>BD Bioscience</td>
			<td>553370</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat IgG Dynabeads</td>
			<td>Invitrogen</td>
			<td>110-35</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagen</td>
			<td>BD Bioscience</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>PBS (1x)</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase, type I</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Cellgro </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich </td>
			<td>F2442 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Endothelial cell growth medium</td>
			<td>(made in lab as described in notes)</td>
			<td></td>
			<td>DMEM containing 20% FBS, 5 &mu;M &beta;-mercaptoethanol, 50 &mu;g/ml ECG, and 1x penicillin/streptomycin</td>
		 </tr>
		 <tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ECGS</td>
			<td>Biomedical Technologies, Inc.</td>
			<td>BT-203</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>384-well culture plate</td>
			<td>Greiner</td>
			<td>T-3037-6</td>
			<td>Plate was prepared with a thin collagen gel, as described in 6; working surface area 10 mm2</td>
		 </tr>
		 <tr>
			<td>Collagen type I</td>
			<td>BD</td>
			<td>354236</td>
			<td>Use at a final concentration of 1.5 mg/ml</td>
		 </tr>
		 <tr>
			<td>M199, 10x</td>
			<td>Invitrogen</td>
			<td>11825-015</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Syto-16</td>
			<td>Invitrogen </td>
			<td>S7578 </td>
			<td>Used as directed in 13 </td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Stereoscopic microscope</td>
			<td>Nikon </td>
			<td>SMZ645</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cell culture incubator</td>
			<td>Thermo </td>
			<td>3110 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>DynaMag</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Table-top centrifuge</td>
			<td>Thermo</td>
			<td>75002430</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

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 offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Using an endothelial-specific antibody that recognizes CD31, endothelial cells were isolated from the ventricles of embryonic (E) 13.5 (Figure 1A) and 18.5 (Figures 1B-1D) embryos. When grown on an untreated culture dish, these cells remain rounded and would form a cobblestone pattern if near confluent (Figure 1A). Dilute collagen has also been used to coat the wells, and while the endothelial cells will adhere to it (Figure 1B), they form fewer chains t...

Watch this Scientific Journal Video about Isolation of Embryonic Ventricular Endothelial Cells at JoVE.com

Isolation of Embryonic Ventricular Endothelial Cells

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

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

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