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In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix

Nature Protocols volume 7, pages 247–255 (2012)Cite this article

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Abstract

We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24–48 h and can be maintained in culture for 5–6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.

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Figure 1
Figure 2: Dissection of the heart from adult zebrafish.
Figure 3: Explant culture of adult zebrafish hearts on fibrin gels.
Figure 4: Epicardial cell outgrowth on fibrin gels.
Figure 5: Proliferation of epicardial cells on fibrin gels.

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Acknowledgements

This work was supported by the American Heart Association (0730214N to C.-L.L.), National Heart, Lung, and Blood Institute Grants (R01HL096121 to C.-L.L. and R01HL096121S1 to C.-L.L. for N.R.), the Wright Foundation (to C.-L.L.), a Research Career Development Award from the Saban Research Institute (C.-L.L.), a National Institute of General Medical Sciences Grant (R01GM055081 to T.-L.T.) and California Institute for Regenerative Medicine (CIRM) postdoctoral fellowships (J.K.). We thank Q. Wu for excellent technical support, E. Fernandez for helping with taking the video and M. Chao for critically reviewing the manuscript prior to submission.

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Author notes

  1. Nicole Rubin and Ying Huang: These authors contributed equally to this work.

Authors and Affiliations

  1. The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California, USA

    Jieun Kim, Nicole Rubin, Ying Huang, Tai-Lan Tuan & Ching-Ling Lien

  2. Division of Cardiothoracic Surgery of Children's Hospital Los Angeles, Los Angeles, California, USA

    Jieun Kim, Nicole Rubin, Ying Huang & Ching-Ling Lien

  3. Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Tai-Lan Tuan & Ching-Ling Lien

Authors
  1. Jieun Kim
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  2. Nicole Rubin
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  3. Ying Huang
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  4. Tai-Lan Tuan
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  5. Ching-Ling Lien
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Contributions

J.K., T.-L.T. and C.-L.L. designed the study; J.K., N.R. and Y.H. carried out the experiments; and J.K., N.R., T.-L.T. and C.-L.L. wrote the manuscript.

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Correspondence to Ching-Ling Lien.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Dissection of zebrafish to collect the heart for culture. (MOV 4391 kb)

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Kim, J., Rubin, N., Huang, Y. et al. In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix. Nat Protoc 7, 247–255 (2012). https://doi.org/10.1038/nprot.2011.440

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