An improved protocol for primary culture of cardiomyocyte from neonatal mice

  • P. Sreejit
  • Suresh Kumar
  • Rama S. Verma


The primary culture of neonatal mice cardiomyocyte model enables researchers to study and understand the morphological, biochemical, and electrophysiological characteristics of the heart, besides being a valuable tool for pharmacological and toxicological studies. Because cardiomyocytes do not proliferate after birth, primary myocardial culture is recalcitrant. The present study describes an improved method for rapid isolation of cardiomyocytes from neonatal mice, as well as the maintenance and propagation of such cultures for the long term. Immunocytochemical and gene expression data also confirmed the presence of several cardiac markers in the beating cells during the long-term culture condition used in this protocol. The whole culture process can be effectively shortened by reducing the enzyme digestion period and the cardiomyocyte enrichment step.


Primary cell culture Neonatal mice Murine cardiomyocyte enrichment Immunostaining Gene expression 



This work is supported by grants to R.S.V. by the Ministry of Human Resource Development (MHRD—BIO/2005–2006/007/MHRD/RAMS/859) and Department of Biotechnology, Ministry of Science and Technology (DBT-BT/PR5392/MED/14/693/2004).

Supplementary material

Supplementary data 1A

Cardiomyocte beating in cluster (MPG 1.15 MB)

Supplementary data 1B

Individual beating cardiomyocytes (MPG 988 kb)

Supplementary data 1C

Single beating cardiomyocyte (MPG 982 kb)

Supplementary data 1D

(MPG 1.42 MB)

11626_2007_9079_MOESM5_ESM.doc (30 kb)
Supplementary Table 1 PCR primers used for the study (DOC 30.5 KB)


  1. Ahuja, P.; Sdek, P.; MacLellan, W.R. Cardiac myocyte cell cycle control in development, disease, and regeneration. Physiol. Rev. 87(2):521–544; 2007.PubMedCrossRefGoogle Scholar
  2. Bahi, N.; Zhang, J.; Llovera, M.; Ballester, M.; Comella, J.X.; Sanchis, D. Switch from caspase-dependent to caspase-independent death during heart development: essential role of endonuclease G in ischemia-induced DNA processing of differentiated cardiomyocytes. J. Biol. Chem. 281(32):22943–22952; 2006.PubMedCrossRefGoogle Scholar
  3. Bick, R.J.; Snuggs, M.B.; Poindexter, B.J.; Buja, L.M.; Van Winkle, W.B. Physical, contractile and calcium handling properties of neonatal cardiac myocytes cultured on different matrices. Cell Adhes. Commun. 6(4)301–310; 1998.PubMedCrossRefGoogle Scholar
  4. Blondel, B.; Roijen, I.; Cheneval, J.P. Heart cells in culture: a simple method for increasing the proportion of myoblasts. Experientia 27:356–358; 1971.PubMedCrossRefGoogle Scholar
  5. Bryja, V.; Bonilla, S.; Cajánek, L.; Parish, C.L.; Schwartz, C.M.; Luo, Y.; et al. An efficient method for the derivation of mouse embryonic stem cells. Stem. Cells 24(4)844–849; 2006.PubMedCrossRefGoogle Scholar
  6. Chlopclkova, Š.; Psotova, J.; Miketova, P. Neonatal rat cardiomyocytes—a model for the study of morphological, biochemical and electrophysiological characteristic of the heart. Biomed. Papers 145:49–55; 2001.Google Scholar
  7. Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162(1):156–159; 1987.PubMedCrossRefGoogle Scholar
  8. Clark, W.J. Selective control of fibroblast proliferation and its effect on cardiac muscle differentiation in vitro. Dev. Biol. 52:263–282; 1976.PubMedCrossRefGoogle Scholar
  9. Desmond, W.J.; Harary I. In vitro studies of beating heart cells in culture. XV. Myosin turnover and the effect of serum. Arch. Biochem. Biophys. 151:285–294; 1972.PubMedCrossRefGoogle Scholar
  10. Duarte, A.I.; Proença, T.; Oliveira, C.R.; Santos, M.S.; Rego C. Insulin restores metabolic function in cultured cortical neurons subjected to oxidative stress. Diabetes 55:2863–2870; 2006.PubMedCrossRefGoogle Scholar
  11. Fioramonti, M.C.; Bryant, J.C.; Mcquilkin, W.T.; Evans, V.J.; Sanford, K.K.; Earle, W.R. The effect of horse serum residue and chemically defined supplements on proliferation of Strain L Clone 929 Cells from the Mouse. Cancer Res. 15(11):763–766; 1955.PubMedGoogle Scholar
  12. Flanders, K.C.; Holder, M.G.; Winokur, T.S. Autoinduction of mRNA and protein expression for transforming growth factor-βs in cultured cardiac cells. J. Mol. Cell Cardiol. 27(2):805–812; 1995.PubMedCrossRefGoogle Scholar
  13. Fu, J.; Gao, J.; Pi, R.; Liu, P. An optimized protocol for culture of cardiomyocyte from neonatal rat. Cytotechnology 49:109–116; 2005.CrossRefGoogle Scholar
  14. Haas, R.; Banerji, S.S.; Culp, L.A. Adhesion site composition of murine fibroblasts cultured on gelatin-coated substrata. J. Cell Physiol. 120(2):117–125; 1984.PubMedCrossRefGoogle Scholar
  15. Harary, I.; Farley, B. In vitro studies on single beating rat heart cells II intercellular communication. Exp. Cell Res. 29:466–474; 1963.PubMedCrossRefGoogle Scholar
  16. Healy, G.M.; Parker, R.C. Cultivation of mammalian cells in defined media with protein and nonprotein supplements. J. Cell Biol. 30(3):539–553; 1966.PubMedCrossRefGoogle Scholar
  17. Kruppenbacher, J.P.; May, T.; Eggers, H.J.; Piper, H.M. Cardiomyocytes of adult mice in long-term culture. Naturwissenschaften 80:132–134; 1993.PubMedCrossRefGoogle Scholar
  18. Limaye, D.A.; Shaikh, Z.A. Cytotoxicity of cadmium and characteristics of its transport in cardiomyocytes. Toxicol. Appl. Pharmacol. 154(1):59–66; 1999.PubMedCrossRefGoogle Scholar
  19. Mark, G.E.; Strasser, F.F. Pacemaker activity and mitosis in cultures of newborn rat heart ventricle cells. Exp. Cell Res. 44:217–233; 1966.PubMedCrossRefGoogle Scholar
  20. Matsuura, K.; Wada, H.; Nagai, T.; Iijima, Y.; Minamino, T.; Sano, M.; et al. Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle. J. Cell Biol. 167(2):351–363; 2004.PubMedCrossRefGoogle Scholar
  21. McKoy, G.; Bicknell, K.A.; Patel, K.; Brooks, G. Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation. Cardiovasc. Res. 74(2):304–312; 2007.PubMedCrossRefGoogle Scholar
  22. Nickson, P.; Toth, A.; Erhardt, P. PUMA is critical for neonatal cardiomyocyte apoptosis induced by endoplasmic reticulum stress. Cardiovasc. Res. 73(1):48–56; 2007.PubMedCrossRefGoogle Scholar
  23. Nuss, H.B.; Marban, E. Electrophysiological properties of neonatal mouse cardiac myocytes in primary culture. J. Physiol. 479(2):265–279; 1994.PubMedGoogle Scholar
  24. Pellieux, C.; Foletti, A.; Peduto, G.; Aubert, J. F.; Nussberger, J.; Beermann, F.; et al. Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2. J. Clin. Invest. 108:1843–1851; 2001.PubMedGoogle Scholar
  25. Polinger, I.S. Separation of cell types in embryonic heart cell cultures. Exp. Cell Res. 63:78–82; 1970.PubMedCrossRefGoogle Scholar
  26. Rao, V.; Merante, F.; Weisel, R.D.; Shirai, T.; Ikonomidis, J.S.; Cohen, G.; et al. Insulin stimulates pyruvate dehydrogenase and protects human ventricular cardiomyocytes from simulated ischemia. J. Thorac. Cardiovasc. Surg. 116(3):485–494; 1998.PubMedCrossRefGoogle Scholar
  27. Remião, F.; Carmo, H.; Carvalho, F.; Bastos, M.L. Cardiotoxicity studies using freshly isolated calcium-tolerant cardiomyocytes from adult rat. In Vitro Cell Dev. Biol.—Animal 37:1–4; 2001.CrossRefGoogle Scholar
  28. Rosenblatt, V.N.; Lepore, M.G.; Cartoni, C.; Beermann, F.; Pedrazzini, T. FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes. J. Clin. Invest. 115(7):1724–1733; 2005.CrossRefGoogle Scholar
  29. Shields, P.P.; Dixon, J.E.; Glembotski, C.C. The secretion of atrial natriuretic factor-(99–126) by cultured cardiac myocytes is regulated by glucocorticoids. J. Biol. Chem. 26:3126–3128; 1988.Google Scholar
  30. Simpson, P.; Savion, S. Differentiation of myocytes in single cell cultures with and without proliferating nonmyocardial cells. Circ. Res. 50:101–116; 1982. Circ. Res. 50:101–116; 1982.PubMedGoogle Scholar
  31. Song, W.; Lu, X.; Feng, Q. Tumor necrosis factor-alpha induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes. Cardiovasc. Res. 45(3):595–602; 2000.PubMedCrossRefGoogle Scholar
  32. Wang, G.W.; Kang, Y.J. Inhibition of doxorubicin toxicity in cultured neonatal mouse cardiomyocytes with elevated metallothionein levels. J. Pharmacol. Exp. Ther. 288(3):938–944; 1999.PubMedGoogle Scholar
  33. Yamashita, N.; Nishida, M.; Hoshida, S.; Kuzuya, T.; Hori, M.; Taniguchi N.; et al. Induction of manganese superoxide dismutase in rat cardiac myocytes increases tolerance to hypoxia 24 h after preconditioning. J. Clin. Invest. 94:2193–2199; 1994.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2008

Authors and Affiliations

  1. 1.Stem Cell and Molecular Biology Laboratory, Department of BiotechnologyIndian Institute of Technology MadrasChennaiIndia
  2. 2.201, Bhupat and Jyoti Mehta School of Biosciences, Department of BiotechnologyIndian Institute of Technology MadrasChennaiIndia

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