We would like to thank Andrea Portbury for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

1. Excising the Embryonic Hearts
<ol>
	<li>Euthanize a timed-pregnant mouse at the desired embryonic day using an approved euthanasia technique. All experiments were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.</li>
	<li>Liberally spray the female with 70% ethanol prior to dissection. Open the female's abdominal cavity to retrieve and excise the uterine horn.</li>
	<li>Place the uterine horn in a Petri dish containing cold 1x phosphate-buffered saline (PBS) and rinse as needed. Cut open the uterine horn via the midline to expose the attached embryonic sacs.</li>
	<li>Cut open an embryonic sac and retrieve an embryo. Place the embryo in a second Petri dish with cold PBS. Return the Petri dish with the uterine horn to ice.</li>
	<li>Decapitate the embryo to improve access to the chest wall. If genotyping is necessary, remove and save the tail in an Eppendorf tube placed on ice.</li>
	<li>With the embryo on its back, cut open the chest wall to visualize the heart and lungs. Depending on the stage, the chest wall may be transparent.</li>
	<li>Carefully lift the heart using forceps and cut the vessels below. Then, cut above the great vessels to free the heart. If necessary, remove extraneous tissues.</li>
	<li>Place the heart in one well of a 24-well dish filled with cold PBS and place the dish on ice.</li>
	<li>Repeat the previous steps until hearts have been excised from all embryos.</li>
</ol>
2. Culture Set-up
<ol>
	<li>In a laminar hood, combine cold Matrigel and the desired culture medium (e.g. 10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM)) at a 1:1 ratio. Do not pre-warm the culture medium.</li>
	<li>Keeping the dilute Matrigel on ice, pipette 500-1,000 &mu;l of the diluted solution into a well of a 24-well culture dish that is also maintained on ice.</li>
	<li>In the hood, carefully pick up and place the excised heart in a well of the Matrigel-containing culture dish while keeping the culture dish on ice.</li>
	<li>If orientation of the embedded heart is crucial: Liberally spray an inverted microscope and the surrounding space with 70% ethanol. In the hood, place the excised heart in a well of the Matrigel-containing culture plate while still on ice. Then, place the plate on a microscope and quickly orient the excised heart as the Matrigel begins to solidify.</li>
	<li>Place the culture dish in a humidified 37 &deg;C incubator (5% CO2) and allow the Matrigel to solidify for approximately 30 min.</li>
	<li>Add 1 ml pre-warmed culture medium to each well containing a heart.</li>
	<li>Hearts can remain in culture for at least four days. Changing the culture medium every two days is recommended.</li>
</ol>
3. Fixation and Visualization
<ol>
	<li>If desired, add a fluorescent live-cell dye (e.g. Syto-16) to visualize the live heart. Photography will be limited to the side of the heart that is visible based on the position in which it was embedded.</li>
	<li>If post-culturing analyses (e.g. whole mount imaging or sectioning) will be performed, remove the culture medium and replace with cold 4% formaldehyde. Place the dish on ice for 30 min.</li>
	<li>After the cold formaldehyde and ice promote dissolution of the Matrigel, replace the fixative (and Matrigel) with fresh formaldehyde and fix hearts overnight at 4 &deg;C.</li>
	<li>Wash the hearts with buffer (e.g. PBS or Tris) and store at 4 &deg;C until further analyses.</li>
</ol>

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The authors declare that they have no competing financial interests.

The current culture system poses significant advantages for embryonic mouse heart studies. This culture system preserves myocardial contractility and the coronary plexus, with limited signs of necrosis, even after four days in culture. Further, the semi-solid matrix provides enough support to maintain the three-dimensional morphology of the developing heart while allowing flexibility to contract and also to hold coated beads in place during culture. Despite this support, this matrix is permeable to fluorescent dye...

Over recent years, the transgenic mouse has been the predominant model system for studying development heart defects. However, other model organisms, such as the zebrafish, have proven to have significant advantages over the mouse. Three major advantages of the zebrafish are the external laying of eggs, for ease of access to the embryos; the optical transparency of the embryos, which allows easy visualization of cardiac development; and the ease of applying small molecular treatments to modulate development of the embryo1. Thus, the development of a culture technique that allowed ex utero growth of an embryonic organ would bypass, at least in part, the limitations currently experienced by researchers studying developmental processes in transgenic mice.
	Ex vivo cardiac culture systems have been developed in both the chick and mouse embryo that allow treatment with small molecules and analysis of how different regions of the heart communicate2-6. For whole mouse heart culture, hearts taken from embryos up to embryonic (E) 12.5 of age can be placed in culture medium with or without rocking2,3,5. Using this technique, embryonic hearts have been successfully incubated to the equivalency of E13.5, and hearts cultured with rocking have been maintained as long as three days (starting at E10.5)3. However, no studies have reported the successful culture of hearts from older embryos. Likewise, rescue experiments have been limited to applying the therapeutic agent globally to the culture medium2.
	A slice culture system, in which hearts are excised, embedded, and sectioned using a vibratome, has also been utilized for both younger hearts, such as E12.5 mouse hearts and Hamburger-Hamilton stage 36 (approximately E16 in the mouse) chick hearts2,4,6, and older hearts, such as post-natal and adult mouse hearts and adult human hearts7,8. While the embryonic analyses have typically utilized 150-&mu;m thick sections2,4, section thickness can be a great as 500 &mu;m without evidence of oxygen deprivation8. These slice cultures have been maintained as long as two months in culture, with most slices maintaining contractility throughout this period9. Compared to studies in isolated cardiomyocytes, these slice cultures allow the co-culture of cardiomyocytes with their neighboring cell types and provide a useful method for ex vivo analysis. However, these cultures require more elaborate set-up than simply placing a heart in culture medium (e.g. embedding the live heart for sectioning on a vibratome), and any analysis is obviously limited to the portion of the heart within the section.
	Given the limitations described above for culturing embryonic mouse hearts and the wealth of transgenic mice available for study, we developed an ex vivo mouse heart culture system similar to an ex vivo lung culture system developed by Weaver, et al.10 Our culture system permits long-term culture and visualization of the remodeling coronary circulation within whole embryonic mouse hearts. In addition, the use of Matrigel allows beads to be held in place near the heart, thus providing a localized treatment with therapeutic agents. These experiments can be performed at different developmental time points to compare the effect of a given treatment on a process such as coronary artery formation. Because small molecules can diffuse through Matrigel, this culture system can also be used to culture dissected regions of the heart near each other to determine whether specific cell-cell contacts are necessary for certain developmental processes or whether paracrine signaling from one region to the other is necessary.
	This culture system is relatively simple and, unlike the slice culture system, makes use of basic culture reagents and set-ups that are readily available in most laboratories. In brief, excised embryonic hearts are cultured in dilute Matrigel, which provides a semi-solid support. This support is sufficient to maintain the three-dimensional morphology of the heart while also allowing the heart to contract. Using this system, whole hearts from older mouse embryos (E14.5-E16.5) can be maintained in culture for up to four days. The entire coronary plexus is maintained, unlike in the slice cultures, so any signaling cues that occur from different regions of the heart remain present. Furthermore, live-cell fluorescent dyes can permeate the Matrigel to allow visualization of the live heart, and protein-conjugated beads can be placed near the heart to provide a localized signaling source. Together, these benefits make this technique an ideal method for studying developmental processes in the embryonic mouse heart.

<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>PBS (1x)</td>
 <td></td>
 <td></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>Growth factor-reduced Matrigel</td>
 <td>BD Bioscience </td>
 <td>356231 </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>24-well culture plate</td>
 <td>Fisher Scientific</td>
 <td>07-200-84</td>
 <td></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>
</tbody>
 </table>

a novel ex vivo culture method for the embryonic mouse heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

Using this technique, the heart maintains its three-dimensional morphology and remains viable, as indicated by continued contractions (Movie 1). These contractions are consistently more prominent in the atria than in the ventricles. Following culture, hearts can be fixed and processed for either immunohistochemistry or histology to examine specific marker expression or structures. Figure 1A shows the base of the ventricles and great arteries of an embryonic mouse heart that was cu...

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A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

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13.2K Views.  University of North Carolina at Chapel Hill. Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.