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Explant culture of adult zebrafish hearts for epicardial regeneration studies

Nature Protocols volume 11, pages 872–881 (2016)Cite this article

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Abstract

Here we describe how to culture adult zebrafish hearts as explants and study the regeneration of epicardial tissue ex vivo, as a means to identify therapeutic targets for heart disease. Uninjured or injured adult hearts are excised, washed and cultured in an incubator with gentle agitation. Heart explants can be prepared within 2 h, and they can be maintained in culture for 30 d or longer. If explants are prepared from appropriate transgenic lines, dynamic behaviors of epicardial cells can be monitored by live imaging using stereofluorescence microscopy. We also describe ex vivo procedures for genetic ablation of the epicardium, cell proliferation assays, tissue grafts and bead grafts. Basic cell culture and surgical skills are required to carry out this protocol. Unlike existing protocols for culturing isolated zebrafish epicardial cells on matrices, procedures described here maintain epicardial cells on an intact cardiac surface, thereby better supporting in vivo cell behaviors. Our protocols complement and extend in vivo studies of heart regeneration.

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Figure 1: Flowchart of experimental procedures.
Figure 2: Heart dissection and explant culture.
Figure 3: Heart chamber and bead grafts.
Figure 4: Ex vivo epicardial regeneration and chamber engraftment.
Figure 5: Sarcomere morphology and expansion of epicardial cells during explant culture.
Figure 6: Spontaneous proliferation of epicardial cells during explant culture.
Figure 7: In situ hybridization on sections of ventricular explants after 2 d of culture.

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Acknowledgements

We thank J. Wang and S. Singh for generating transgenic fish; J. Burris, N. Lee, A. Dunlap, S. Davies, T. Thoren and N. Benkaci for fish care; A.L. Dickson for artwork; and A. Shoffner for comments on the manuscript. This work was funded by a postdoctoral fellowship from the American Heart Association to J.C. and grants from the US National Institutes of Health (NIH; R01 HL081674 and R01 HL131319) to K.D.P.

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  1. Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA

    Jingli Cao & Kenneth D Poss

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  1. Jingli Cao
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  2. Kenneth D Poss
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J.C. and K.D.P. designed the study and wrote the manuscript. J.C. performed all experiments.

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Correspondence to Kenneth D Poss.

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The authors declare no competing financial interests.

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Cao, J., Poss, K. Explant culture of adult zebrafish hearts for epicardial regeneration studies. Nat Protoc 11, 872–881 (2016). https://doi.org/10.1038/nprot.2016.049

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