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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Published: May 24th, 2013

DOI:

10.3791/50359

1McAllister Heart Institute, 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.

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.

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,....

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1. Excising the Embryonic Hearts

  1. 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.
  2. Liberally spray the female with 70% ethanol prior to dissection. Open the female's abdominal cavity to retrieve and excise the uterine horn.
  3. Place the uterine horn in a Petri dish containing cold 1x phosphate-buffered saline (.......

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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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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.......

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

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Name Company Catalog Number Comments
REAGENTS
Timed-pregnant mice To be dissected at the embryonic stage of interest
PBS (1x)
DMEM Cellgro
FBS Sigma-Aldrich F2442
Growth factor-reduced Matrigel BD Bioscience 356231
Syto-16 Invitrogen S7578 Used as directed in 13
24-well culture plate Fisher Scientific 07-200-84
EQUIPMENT
Stereoscopic microscope Nikon SMZ645
Cell culture incubator Thermo 3110

  1. Tu, S., Chi, N. C. Zebrafish models in cardiac development and congenital heart birth defects. Differentiation. 84, 4-16 (2012).
  2. Lavine, K. J., et al. Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development. Genes Dev. 20, 1651-1666 (2006).
  3. Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, M. A. Coronary arteries form by developmental reprogramming of venous cells. Nature. 464, 549-553 (2010).
  4. Stuckmann, I., Evans, S., Lassar, A. B. Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation. Dev. Biol. 255, 334-349 (2003).
  5. Zhang, J., et al. The FGF-BMP signaling axis regulates outflow tract valve primordium formation by promoting cushion neural crest cell differentiation. Circ. Res. 107, 1209-1219 (2010).
  6. Pillekamp, F., et al. Establishment and characterization of a mouse embryonic heart slice preparation. Cellular Physiology and Biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology. 16, 127-132 (2005).
  7. Habeler, W., Peschanski, M., Monville, C. Organotypic heart slices for cell transplantation and physiological studies. Organogenesis. 5, 62-66 (2009).
  8. Janssen, P. M., Lehnart, S. E., Prestle, J., Hasenfuss, G. Preservation of contractile characteristics of human myocardium in multi-day cell culture. J. Mol. Cell Cardiol. 31, 1419-1427 (1999).
  9. Brandenburger, M., et al. Organotypic slice culture from human adult ventricular myocardium. Cardiovasc. Res. 93, 50-59 (2012).
  10. Weaver, M., Dunn, N. R., Hogan, B. L. Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis. Development. , 127-2695 (2000).
  11. Viragh, S., Challice, C. E. The origin of the epicardium and the embryonic myocardial circulation in the mouse. Anat. Rec. 201, 157-168 (1981).
  12. Kirby, M. L. . Cardiac Development. , (2007).
  13. Arima, S., et al. Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement. Development. 138, 4763-4776 (2011).

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