Isolation and Culture of Neonatal Mouse Cardiomyocytes

Summary

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.

Abstract

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.

Introduction

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

Protocol

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 rat neonatal cardiomyocytes. Scale media/enzyme usage as appropriate.

For work with neonatal rodents, refer to your local ....

Results

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 resulti.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	cellgro	25-053-CI
1x Penicillin/ Streptomycin solution with EDTA in HBSS	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	cellgro	21-022-CV
DMEM high glucose	cellgro	10-013-CV
M-199	cellgro	10-060-CV
fetal bovine serum	cellgro	35-011-CV	cell-culture grade
horse serum	cellgro	35-030-CV	cell-culture grade
Leibovitz L-155	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	Fisherbrand	22363548	appropriate filter size:40 μm-100 μm
syringe filter 0.2 μm	Fisherbrand	09-719C	for sterile filtration of digestion medium
straight scissors	Fine Sciences Tools	91460-11
curved scissors	Fine Science Tools	91461-11
Dumont No. 7 forceps	Fine Science Tools	91197-00
perforated spoon	Fine Science Tools	10370-19	optional, for transfer of heart tissue
Trypan blue	Gibco	15250-061	live cell staining
Neubauer hemocytometer	Prosource Scientific	3500	alternatively use: disposable hemocytometer C-chip or automated cell counting systems
50 ml Falcon tubes	Fisherbrand	14-432-23
15 ml Falcon tubes	Fisherbrand	05-527-90
20 ml syringe	BD Medical	14-820-19
10 ml serological pipette	Falcon	357551
30 mm cell culture dish	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	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.