Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes.
Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.
Cardiovascular Research Institute, University of California, San Francisco.
Justin
J
Judd
Cardiovascular Research Institute, University of California, San Francisco.
Jonathan
J
Lovas
Cardiovascular Research Institute, University of California, San Francisco; Guo.Huang@ucsf.edu.
Guo N
GN
Huang
Cell Biochem Biophys. 2011 Sep;61(1):93-101
21327944
In Vitro Cell Dev Biol. 1990 May;26(5):464-70
2351640
J Cell Sci. 2004 Jul 1;117(Pt 15):3295-306
15226401
J Cell Mol Med. 2014 Aug;18(8):1509-18
24981391
Open Biol. 2015 Jun;5(6):150038
26085516
Nature. 2010 Mar 25;464(7288):606-9
20336145
Proc Natl Acad Sci U S A. 2006 Oct 17;103(42):15546-51
17032753
Genes Dev. 2005 May 15;19(10):1175-87
15870258
PLoS One. 2015 Feb 11;10(2):e0116068
25671422
Nat Protoc. 2007;2(5):1236-47
17546019
J Biochem. 1989 Apr;105(4):638-43
2527229
J Cardiovasc Pharmacol Ther. 2015 Jan;20(1):104-12
24917562
J Vis Exp. 2014 Jun 24;(88):e51666
24998400
Biomaterials. 2015 Aug;61:257-65
26005764
J Biol Chem. 1992 May 5;267(13):9269-74
1374390
Elife. 2015 Aug 06;4:null
26247711
J Vis Exp. 2013 Sep 06;(79):null
24056408
J Gene Med. 2003 Sep;5(9):765-72
12950067
Sci Rep. 2015 Mar 09;5:8883
25748532
J Mol Cell Cardiol. 1982 Jul;14(7):397-412
7175947
Cardiovasc Res. 2000 Jan 1;45(1):123-7
10728324
Nat Med. 2007 Aug;13(8):970-4
17660827
Am J Physiol. 1996 Sep;271(3 Pt 2):H1250-5
8853365
Circ Res. 1987 Oct;61(4):560-9
3652400
Am J Physiol. 1996 Nov;271(5 Pt 2):H2183-9
8945939
J Cell Physiol. 2005 Mar;202(3):891-9
15389635
Development. 2013 Dec;140(23):4683-90
24255096
Circ Res. 2001 Jul 6;89(1):20-5
11440973
Circ Res. 2015 Jan 2;116(1):35-45
25249570
Am J Pathol. 1979 Nov;97(2):411-32
525677
J Biol Chem. 2011 Mar 11;286(10):8644-54
21209082
J Physiol Sci. 2007 Dec;57(6):327-35
17980092
Biotechnol Prog. 2006 Sep-Oct;22(5):1294-300
17022666
Naturwissenschaften. 1993 Mar;80(3):132-4
8464521
Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):E1848-57
22645348
J Vis Exp. 2014 May 21;(87):null
24894542
Biochem Biophys Res Commun. 1985 Apr 16;128(1):376-82
3985976
PLoS One. 2010 Sep 03;5(9):e12559
20838637
Nature. 2010 Mar 25;464(7288):601-5
20336144
J Clin Invest. 1997 Jun 1;99(11):2644-54
9169494
Dev Cell. 2009 Feb;16(2):233-44
19217425
Am J Physiol Heart Circ Physiol. 2000 Jul;279(1):H429-36
10899083
Science. 2011 Feb 25;331(6020):1078-80
21350179
Sci Transl Med. 2015 Mar 18;7(279):279ra38
25787764
Nature. 2013 May 9;497(7448):249-253
23594737
Dev Biol. 2012 May 15;365(2):339-49
22426105
Circulation. 2000 Feb 15;101(6):660-7
10673259