title: Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts.
abstract: This protocol describes how to isolate primary cardiomyocytes from adult zebrafish hearts and culture them for up to 4 weeks, thereby using them as an alternative to in vivo experiments. After collagenase digestion of the ventricle, cells are exposed to increasing calcium concentrations in order to obtain high-purity cardiomyocytes. The whole isolation process can be accomplished in 4-5 h. The culture conditions we established allow the cells to preserve their mature sarcomeric integrity and contractile properties. Furthermore, adult zebrafish cardiomyocytes in culture, similarly to zebrafish in vivo heart regeneration, undergo partial dedifferentiation and, in contrast to their mammalian counterparts, are able to proliferate. Our protocol enables the study of structural and functional properties in close-to-native cardiomyocytes and allows the application of in vitro techniques and assays that are not feasible to perform in living animals.
authors.affiliation: Center of Regenerative Medicine in Barcelona, Barcelona, Spain.
authors.forename: Veronika
authors.initials: V
authors.lastname: Sander
authors.forename: Guillermo
authors.initials: G
authors.lastname: Suñe
authors.forename: Chris
authors.initials: C
authors.lastname: Jopling
authors.forename: Cristina
authors.initials: C
authors.lastname: Morera
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Izpisua Belmonte
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D018929:Cell Culture Techniques; D054337:Cell Dedifferentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D003470:Culture Media; D005337:Fibrin / Q000737:chemistry; D032383:Myocytes, Cardiac / Q000166:cytology*; D015027:Zebrafish
chemical_list: D003470:Culture Media; D005337:Fibrin; D002118:Calcium
references.citation: Br J Exp Pathol. 1979 Feb;60(1):96-106
references.pmid: 465309
references.citation: Cold Spring Harb Protoc. 2012 Aug 01;2012(8):
references.pmid: 22854565
references.citation: BMC Mol Biol. 2008 Nov 12;9:102
references.pmid: 19014500
references.citation: Physiol Rev. 2003 Jan;83(1):59-115
references.pmid: 12506127
references.citation: Heart. 2011 Jan;97(1):15-9
references.pmid: 20884787
references.citation: Dev Dyn. 2003 Sep;228(1):30-40
references.pmid: 12950077
references.citation: Biochem Biophys Res Commun. 2008 Sep 12;374(1):143-6
references.pmid: 18602892
references.citation: Cardiovasc Res. 2012 Mar 1;93(3):516-24
references.pmid: 22200588
references.citation: Circ Res. 1982 Aug;51(2):117-30
references.pmid: 7094224
references.citation: Eur Heart J. 2002 Jun;23(11):849-57
references.pmid: 12042006
references.citation: Mol Vis. 2008 May 19;14:951-63
references.pmid: 18509551
references.citation: Dev Cell. 2011 Mar 15;20(3):397-404
references.pmid: 21397850
references.citation: Am J Physiol Heart Circ Physiol. 2001 Oct;281(4):H1711-9
references.pmid: 11557562
references.citation: Science. 2002 Dec 13;298(5601):2188-90
references.pmid: 12481136
references.citation: Drug Discov Today Dis Models. 2007;4(4):219-225
references.pmid: 19081827
references.citation: Development. 2011 Aug;138(16):3421-30
references.pmid: 21752928
references.citation: Cardiovasc Res. 1998 Aug;39(2):280-300
references.pmid: 9798514
references.citation: Nat Protoc. 2012 Mar 29;7(4):782-8
references.pmid: 22461067
references.citation: Am J Physiol Regul Integr Comp Physiol. 2002 Apr;282(4):R1200-9
references.pmid: 11893626
references.citation: Science. 2011 Feb 25;331(6020):1078-80
references.pmid: 21350179
references.citation: Bioessays. 2011 Sep;33(9):669-73
references.pmid: 21748752
references.citation: Cell. 2009 Jul 23;138(2):257-70
references.pmid: 19632177
references.citation: Nature. 2010 Mar 25;464(7288):601-5
references.pmid: 20336144
references.citation: Methods Mol Biol. 2010;633:113-24
references.pmid: 20204624
references.citation: J Mol Cell Cardiol. 2011 Sep;51(3):288-98
references.pmid: 21723873
references.citation: Cell Physiol Biochem. 2011;27(1):1-12
references.pmid: 21325816
references.citation: Nat Protoc. 2012 Jan 19;7(2):247-55
references.pmid: 22262006
references.citation: Cell Cycle. 2012 Mar 15;11(6):1195-201
references.pmid: 22391208
references.citation: J Clin Invest. 1995 Sep;96(3):1280-7
references.pmid: 7657802
references.citation: Genes Dev. 2005 May 15;19(10):1175-87
references.pmid: 15870258
references.citation: Nature. 2010 Mar 25;464(7288):606-9
references.pmid: 20336145
references.citation: Nat Rev Mol Cell Biol. 2011 Feb;12(2):79-89
references.pmid: 21252997
references.citation: Am J Physiol Regul Integr Comp Physiol. 2011 Jan;300(1):R56-66
references.pmid: 20926764
references.citation: Circulation. 2012 Dec 18;126(25):3017-27
references.pmid: 23151342