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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Published: September 6th, 2013

DOI:

10.3791/50154

1Randall and Cardiovascular Divisions, King’s College London, 2Division of Cardiology, School of Medicine, University of California San Diego

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.

The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

The earliest reports for the successful dissociation and culture of rodent heart cells dates back to the 1960's 1,2. Even then, Harary and Farley noticed that cultured cardiomyocytes "may provide a unique system for the study of the requirements of the periodic contractility [, and may] provide a means of determining the contribution of various metabolic pathways for the [beating] process". Although Harary and Farley isolated and cultured cardiomyocytes from young rats, and the original protocol has been adapted and modified by many scientists over the years, the general isolation and culturing procedure has not greatly changed. However, better enzymes

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The following procedure describes a two-day protocol 16,17 for the isolation and culture of neonatal mouse cardiomyocytes. All solutions are sterile or sterile filtered. All tools are sterilized by surface sterilization with 75% ethanol. Except for the initial tissue extraction, all steps are performed in a sterile laminar flow cell culture hood. This protocol is intended for the isolation of neonatal mouse hearts from one-two litter(s) - approximately 5-14 pups, but may be adapted for larger litter sizes and .......

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Using this protocol, we isolated hearts from 8 one day old neonatal mice (Figures 1A, 1B, Supplemental Movie S1). After washing and mincing the hearts with scissors (Figures 1C-1F), tissue fragments were predigested in isolation medium over night at 4 °C with gentle agitation. Following predigestion (Figure 1G), we transferred the tissue fragments into freshly made digestion medium, and incubated the tissue fragments for 20 min at 37 °C with gentle agitation. The.......

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The use of animal models to study cardiac diseases has become standard in cardiovascular research. Closer biochemical characterization of these models (i.e. studying direct responses of cardiac cells to biochemical or biomechanical stimuli) typically requires the isolation of heart tissues or cardiomyocytes. Studies investigating physiological responses of the heart ex vivo (e.g. to acetylcholine 40, or in ischemia-reperfusion scenarios 41) generally utilize langendorff-pe.......

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We are grateful to Prof. emeritus Jean-Claude Perriard and Evelyn Perriard (Swiss Federal Institute of Technology, Switzerland) for the introduction into isolation techniques for neonatal rat and mouse cardiomyocytes. We would like to thank Prof. Ju Chen and Prof. Sylvia Evans (UCSD, USA) for their support. Work in the laboratory of EE was funded by an MRC Career Establishment Grant. SL is supported by a K99/R00 pathway to independence award from the NIH/NHLBI (HL107744). TMM was supported by a postdoctoral fellowship from the American Heart Association (11POST7310066).

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
BDM (2,3-Butanedione monoxime) 6-8 Sigma B-0753 prepare 0.2M stock solution in HBSS (without Ca2+, Mg2+), filter sterilize, can be kept at 4 °C up to 6 months; Caution: Prolonged usage of BDM other than during isolation procedure may result in non-beating cells, decreased cell viability and/or significantly altered gene-expression during cardiomyocyte culture46,47.
Collagenase/Dispase Roche 10269638001 can be substituted with collagenase type II from Worthington
Collagenase type II3 Worthington CLS-2 substitute for Collagenase/Dispase mix from Roche
1x trypsin solution (0.25%) with EDTA e.g. cellgro 25-053-CI  
1x Penicillin/ Streptomycin solution with EDTA in HBSS e.g. cellgro 30-002-Cl  
1x PBS (without Ca2+, Mg2+) e,g, cellgro 21-040-CV  
HBSS (Hank's balanced salt solution; without Ca2+, Mg2+) 4 e.g. cellgro 21-022-CV  
DMEM high glucose e.g. cellgro 10-013-CV  
M-199 e.g. cellgro 10-060-CV  
fetal bovine serum e.g. cellgro 35-011-CV cell-culture grade
horse serum e.g. cellgro 35-030-CV cell-culture grade
Leibovitz L-155 e.g. cellgro 10-045-CV  
AraC (Cytosine-B-D-arabino-furanoside hydrochloride) Sigma C-6645 proliferation inhibitor, prepare 1 mM stock solution in H2O, filter sterilize, store at 4 °C
phenylephrine Sigma P-6126 chronotropic agent, prepare 100 mM stock solution in H2O, filter sterilize, store at 4 °C or -20 °C
isoproterenol hydrochloride Sigma I-6501 chronotropic agent, prepare 1 mM stock solution in H2O, filter sterilize, store at 4 °C or -20 °C
0.1% collagen solution Sigma C-8919 extracellular matrix for coating
3 mg/ml collagen type 1 solution Advanced BioMatrix 5005-B alternative to Sigma collagen solution
cell strainer e.g. Fisherbrand 22363548 appropriate filter size:40 μm-100 μm
syringe filter 0.2 μm e.g. Fisherbrand 09-719C for sterile filtration of digestion medium
straight scissors e.g. Fine Sciences Tools 91460-11  
curved scissors e.g. Fine Science Tools 91461-11  
Dumont No. 7 forceps e.g. Fine Science Tools 91197-00  
perforated spoon e.g. Fine Science Tools 10370-19 optional, for transfer of heart tissue
Trypan blue e.g. Gibco 15250-061 live cell staining
Neubauer hemocytometer e.g. Prosource Scientific 3500 alternatively use: disposable hemocytometer C-chip or automated cell counting systems
50 ml Falcon tubes e.g. Fisherbrand 14-432-23  
15 ml Falcon tubes e.g. Fisherbrand 05-527-90  
20 ml syringe e.g. BD Medical 14-820-19  
10 ml serological pipette e.g. Falcon 357551  
30 mm cell culture dish e.g. Nunc 153066 for standard culture of cardiomyocytes
30 mm cell culture dish, glass bottom MatTek P35G-0-10-C for live cell imaging with inverted microscope
10 cm cell culture dish e.g. Nunc 172958 for preplating
Escort III Sigma L3037 for liposomal transfection, alternatively use lipofectamin 2000
Lipofectamine 2000 Life Technologies, Invitrogen 52887 substitute for Escort III
      Buffers and media:
  • Isolation medium (filter sterilize)
    20 mM BDM
    0.0125% trypsin
    in HBSS4 (without Ca2+, Mg2+)
  • Digestion medium (filter sterilize)
    20 mM BDM
    1.5 mg/ml Roche Collagenase/Dispase enzyme mix
    in L15 medium
  • Plating medium
    65% DMEM high glucose
    19% M-199
    10% horse serum
    5% fetal calf serum
    1% penicillin/streptomycin
  • Maintenance medium
    78% DMEM high glucose
    17% M-199
    4% horse serum
    1% penicillin/streptomycin
    optional:
    1 μM AraC
    1 μM isoproterenol or 0.1 mM phenylephrine
The plating and maintenance medium can be stored at 4 °C for up to 6 months.

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