In Vitro Cell.Dev.Biol.
—Animal (2008) 44:45–50
DOI 10.1007/s11626-007-9079-4
An improved protocol for primary culture of cardiomyocyte
from neonatal mice
P. Sreejit & Suresh Kumar & Rama S. Verma
Received: 13 September 2007 / Accepted: 11 December 2007 / Published online: 23 February 2008 / Editor: J. Denry Sato.
# The Society for In Vitro Biology 2008
Abstract The primary culture of neonatal mice cardiomyo-
cyte model enables researchers to study and understand the
morphological, biochemical, and electrophysiological char-
acteristics of the heart, besides being a valuable tool for
pharmacological and toxicological studies. Because cardio-
myocytes 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.
Keywords Primary cell culture . Neonatal mice .
Murine cardiomyocyte enrichment . Immunostaining .
Gene expression
Electronic supplementary material The online version of this article
(doi:10.1007/s11626-007-9079-4) contains supplementary material,
which is available to authorized users.
P. Sreejit : S. Kumar : R. S. Verma
Stem Cell and Molecular Biology Laboratory,
Department of Biotechnology,
Indian Institute of Technology Madras,
Chennai 600036 TN, India
R. S. Verma (*)
201, Bhupat and Jyoti Mehta School of Biosciences,
Department of Biotechnology,
Indian Institute of Technology Madras,
Chennai 600 036 TN, India
e-mail: vermars@iitm.ac.in
The neonatal murine cardiomyocyte culture was first
described by Harary and Farley (1963); since then, several
modifications have been developed and reported in the past
40 yr. Various groups working with adult murine cardio-
myocyte cultures have elaborated on issues like finding and
formulating conditions for proper tissue dissociation such
as enzymes and duration of enzyme treatment, in selecting
various growth supplements and appropriate serum con-
centrations for cell attachment, and also appropriate
attachment surfaces especially for long-term cell cultures
(Kruppenbacher et al. 1993; Chlopclkova et al. 2001).
Neonatal murine cardiomyocyte cultures have been used as
a model for studying contraction, ischemia, and hypoxia at
the cellular level (Yamashita et al. 1994; Bahi et al. 2006).
Murine cardiomyocyte cultures have also been used to
study the toxicology of drugs and their transport (Limaye
and Shaikh 1999; Wang and Kang 1999).
In this report, we describe a simple and rapid method for
the isolation of cardiomyocytes from neonatal mice heart
and their maintenance in primary cultures that consistently
yielded long-term cardiomyocyte cultures in our laboratory.
This protocol does not involves the addition of growth
supplements like carnitine, creatinine, taurine, and non-
muscle cells proliferation inhibitors such as 9-β-D-arabino-
furanosyl cytosine and 5-bromo-2′-deoxyuridine or by
increasing the Ca2+ concentration in the washing and
incubation mediums (Kruppenbacher et al. 1993; Nuss
and Marban 1994; Remião et al. 2001; Fu et al. 2005). This
protocol reduces the processing time considerably. For
culturing the neonatal cardiomyocytes in vitro, cardiomyo-
cyte enrichment and the enzyme digestion steps are the two
critical steps. The cardiomyocyte enrichment step, which
removes nonmuscle cells, is crucial for obtaining a homog-
enous yield of myocytes for culture. Several techniques,
including the differential attachment technique (Polinger
46 SREEJIT ET AL.

Table 1. Comparison of various protocols used for cultivation of neonatal mouse cardiomyocytes

Research Ingredient variations Protocol variations

group
(year of Media and supplements Enzyme used Supplements/ Enzyme treatment and Cardiomyocyte
publication) used inhibitors number of repeats enrichment

Our study DMEM/F12 (1:1) with 0.5% trypsin EDTA (~1 ml of 2 mM L-glutamine, 37° C/4 min in a water bath Differential
(2007) in fetal calf serum (20%) trypsin for every 100 mg of 0.1 mM nonessential with stirring and intermittent attachment
BALB/c and horse serum (5%) tissue) amino acids, 3 mM pipeting; solution allowed to technique at
mice with antibiotics sodium pyruvate stand for 1 min; supernatant 37° C for 2–
and bovine insulin collected into 15-ml falcon 3h
(1 μg/ml) tube on ice, to which 2 ml
media supplemented with
20% fetal calf serum was
added; digested suspensions
were pooled and centrifuged
at 3,500 rpm/10 min/4° C.
Repeated three to five times
till tissue is digested
completely
Wang and MEM with 20% FBS 0.25% trypsin in HBSS – 37° C /15 min; centrifuged at Differential
Kang with antibiotics (without Ca++ & Mg++) at 200×g/8 min repeated till the attachment
(1999) in pH 7.4 tissue is completely dissolved technique at
FVB 37° C for 2 h
mice
Song et al. M199 with 10% fetal calf 0.625 mg/ml collagenase type 5 mM D-glucose 37° C /40 min; filtered through Differential
(2000) in serum II a polypropylene macroporous attachment
C57BL6 filter (mesh opening technique for
and iNOS 105 μm); centrifuged at 40 min
(2/2) 1,200 rpm/5 min
mutant
mice
Nuss and DMEM (nutrient mixture 0.25 mg/ml collagenase in – 37° C with slow rotation of the –
Marban F-12 HAM) with 10% Joklik MEM with following tube containing the digestion
(1994) in fetal bovine serum additions (mM unless solution; supernatant during
CD-1 otherwise given): HEPES, 10; repeated collagenase
mice sodium pyruvate, 10; L- digestions pooled to digestion
glutamine, 5; nicotinamide, solution (without
1; L-ascorbate, 0.4; collagenase) on ice; fractions
adenosine, 1; D-ribose, 1; centrifuged at low speed;
MgCl2, 1; taurine, 1; DL- resuspended in digestion
carnitine, 2; potassium solution with 0.2% fetal
bicarbonate, 26; CaCl2, 0.5; bovine serum; filtered with
gentamycin, 10 μg/ml cell strainer, repeated
Rosenblatt 3:1 mix of DMEM and 0.45 mg/ml collagenase and – – Differential
et al. M 199 with 10% horse 1 mg/ml pancreatin in buffer attachment
(2005) in serum and 5% fetal technique for
C57BL/6 bovine serum with 45 min
mice antibiotics
Nickson et 4:1 mix of DMEM:M199 0.03% Collagenase type II and 10 mM glutamine Enzyme treatment for 20 min Two rounds of
al. (2007) with 10% heat- 0.06% pancreatin in a and 0.2 mM at 37° C; supernatant differential
in wild- inactivated horse serum balanced salt solution bromodeoxyuridine collected and cells isolated attachment
type and and 5% heat-inactivated containing MgSO4 7H20 by centrifugation at technique for
Puma fetal bovine serum with (0.2 g/l), NaCl (6.8 g/l), KCl 1,400 rpm/4 min; 2h
knockout antibiotics (0.4 g/l), NaH2PO4H2O resuspension in 2 ml of
mice (1.5 g/l), glucose (1 g/l), and newborn calf serum at
HEPES (4.76 g/l) at pH 7.5 37° C; repeated four times
 IMPROVED PROTOCOL FOR CULTURE OF NEONATAL MICE CARDIOMYOCYTE
1970; Blondel et al. 1971), deprivation of serum (Shields
et al. 1988), density gradient centrifugation (Flanders et al.
1995), radiation (Desmond and Harary 1972), glutamine
deprivation (Clark 1976), and use of chemical reagents to
inhibit nonmyocyte proliferation (Simpson and Savion
1982), have been used to culture of neonatal murine
cardiomyocytes. The enzyme digestion step for dissociating
cells from heart tissue too is critical during culturing of
neonatal cardiomyocytes. It has been reported that short
repetitive trypsinization of heart tissue helps in obtaining a
higher yield of undamaged myocytes, rather than a single
digestion for longer period (Mark and Strasser 1966). The
incubation period with proteolytic enzyme depends on the
amount of intact tissue left during dissociation into single
cells. Although repetitive digestion of heart tissue with
proteolytic enzymes gives good yield, the cell viability
decreases if the tissue is overexposed to proteolytic
enzymes. Cardiomyocytes lose their ability to proliferate
shortly after birth, and growth of heart tissue is governed by
cell growth rather than by proliferation (Ahuja et al. 2007;
Figure 1. Phase-contrast photo-
micrograph of cultures showing
different morphologies of beat-
ing cardiomyocytes in culture
(a, b, c, d; magnification: ×400).
Supplementary data—
videographs 1a, 1b, 1c, 1d.
47
McKoy et al. 2007). Because both mice and rats belong to
the murine family, protocols for neonatal mice cardiomyo-
cyte culture used by many groups are much similar to those
used with neonatal rats (Wang and Kang 1999; Song et al.
2000; Pellieux et al. 2001; Matsuura et al. 2004; Rosenblatt
et al. 2005; Nickson et al. 2007). A comparison of the
protocols is shown along with the report (Table 1). The
present report describes a convenient, reliable, and time-
saving protocol, which has been successfully demonstrated
for consistent cardiomyocyte cultures. It involves decreas-
ing the duration of enzymatic digestion to a maximum of
3 h during which nonmyocytes will be removed too.
BALB/c neonatal and adult mice were procured from
Tamil Nadu Veterinary and Animal Sciences University,
Chennai. All procedures, approved by the Institutional
Animal Ethics Committee (IIT Madras, India) and the
Committee for the Purpose of Control and Supervision of
Experiments on Animals, Government of India, were
performed under the Rule 5(a) of the Breeding and
Experiments on Animals (Control and Supervision) Rules
48 SREEJIT ET AL.

from the heart with a sterile forceps, to prevent clot

Figure 2. Immunocytochemical analysis of GATA-4 and Nkx-2.5 in
cardiomyocytes (double staining). (a) Phase-contrast photomicrograph
of culture. (b) Positive staining with anti-GATA-4 antibody. (c) Positive
staining with anti-Nkx-2.5 antibody (original magnification, ×200).
(1998). Neonatal mice 1–3-d-old were killed by cervical
dislocation. Whole hearts were excised and immediately
transferred into ice-cold phosphate-buffered saline (PBS;
Ca2+ and Mg2+ free). The blood was gently squeezed out
formation inside the lumen of heart. The excised hearts were
again washed with chilled PBS and followed by sterile ice-
cold balanced salt solution (20 mM hydroxyethylpiperazi-
neethanesulfonic acid–NaOH [pH 7.6], 130 mM NaCl,
1 mM NaH2PO4, 4 mM glucose, 3 mM KCl), in which
the tissue was kept for 10 min. The ventricles were excised
in a sterile 60-mm Petri dish, and the auricles were carefully
removed and discarded. The tissues were then minced with
a sterile scalpel blade into small pieces less than or equal to
1 mm3 in 0.05% trypsin ethylenediamine tetraacetic acid
(EDTA; Invitrogen, Carlsbad, CA; 0.1 ml per heart) and
transferred into sterile 15-ml falcon tubes. The myocardial
cells were dispersed by incubating with 0.5% Trypsin
EDTA (~1 ml of Trypsin for every 100 mg of tissue), which
was then mixed by intermittent pipeting along with stirring
at 37° C in a water bath for 4 min. The cell suspension was
allowed to stand for 1 min. The supernatant containing
single cells was collected into a 15-ml falcon tube kept on
ice, to which 2 ml Dulbecco’s modified Eagle’s medium
(DMEM)/F12 (1:1) medium (Gibco, Carlsbad, CA) supple-
mented with 20% fetal calf serum (Gibco) was added, and
the digestion step was repeated three times. The cell
suspensions from each digestion was pooled and centri-
fuged at 3,500 rpm for 10 min at 4° C.
The cell pellet was resuspended in a maintenance
medium containing DMEM/F12 (1:1) medium supple-
mented with fetal calf serum (20%), horse serum (Gibco)
(5%), penicillin (100 U/ml), and streptomycin (100 mg/ml;
Cambrex, Verviers, Belgium), 2 mM L-glutamine (Cam-
brex), 0.1 mM nonessential amino acids (Gibco), 3 mM
sodium pyruvate (Gibco), and bovine insulin (1 μg/ml;
USV, India). Viability of cardiomyocytes was assessed by
Trypan blue exclusion test.
The cells were plated on plates precoated with 1%
gelatin and incubated in 95% air and 5% CO2 at 37° C for
~2–3 h, to allow the differential attachment of nonmyo-
cardial cells. The nonadhesive cells (cardiomyocytes) were

Figure 3. Reverse transcrip-

tion-polymerase chain reaction
(RT-PCR) analysis for cardiac
transcription factors (Nkx-2.5,
GATA-4, and MEF-2C), as well
as cardiac structural and func-
tional proteins (α-Cardiac Actin,
Cardiac Troponin T, Connexin
43) in cardiomyocytes on dif-
ferent days of primary culture.
Adult mouse heart was used as a
positive control, and β-actin
was used as a loading internal
control.
 IMPROVED PROTOCOL FOR CULTURE OF NEONATAL MICE CARDIOMYOCYTE
transferred into a sterile tube. The viability of cells (85–
90%) was assessed by the Trypan blue exclusion test. After
counting, the myocyte-enriched suspension was plated on to
culture dishes at a density of 2×104 cells per cm2. The cells
were incubated in a humid 5% CO2 incubator at 37° C. The
Medium was replenished after 72 h and there after every
48 h. Beating myocardial cells were clearly observed on
third day in culture (Fig. 1) through an inverted microscope
(Nikon Eclipse TS100, Melville, NY), and video graphs of
beating cells were recorded with a digital camera (Nikon
Coolpix5400; supplementary data—videographs 1a, 1b, 1c,
1d). Cells were maintained up to 4 wk for further study.
To demonstrate that cultured cells are cardiomyocytes, we
characterized beating cells by immunocytochemistry using
primary antibodies: mouse monoclonal anti-GATA-4 (1:100;
Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit
polyclonal anti-Nkx-2.5 (1:100; Santa Cruz Biotechnology)
and appropriate secondary antibodies: R-Phycoerythrin-
labelled goat anti-mouse IgG (1:300; Sigma, St. Louis,
MO) and fluorescein isothiocyanate-labeled goat anti-rabbit
IgG (1:300; Sigma). The immunofluorescent staining of cells
showed the presence of cardiac-specific proteins; the cells
were stained positive for GATA-4 (Fig. 2b) and Nkx-2.5
(Fig. 2c) in the nucleus.
Furthermore, we have also analyzed the time course
expression of cardiomyocyte-specific transcription factors
and structural and functional proteins by reverse transcrip-
tase–polymerase chain reaction (RT-PCR). Total ribonu-
cleic acid (RNA) from cardiomyocytes on different days of
primary culture and adult mouse heart were isolated by
using the acid guanidinium thiocyanate–phenol–chloroform
extraction method (Chomczynski and Sacchi 1987). RNA
was subjected to RT-PCR by using M-MLV Reverse
Transcriptase (New England Biologicals, Beverly, MA).
Primers are as follows: Nkx-2.5, GATA-4, MEF-2C, α-
Cardiac Actin, Cardiac Troponin T, Connexin 43 (Supple-
mentary Table 1). RT-PCR results revealed that cells
expressed cardiac transcription factors (Nkx-2.5, GATA-4,
and MEF-2C), as well as cardiac structural and functional
proteins (α-Cardiac Actin, Cardiac Troponin T, Connexin
43; Fig. 3). Thus, our immunocytochemical and gene
expression data confirmed the presence of several cardiac
markers in the beating cells.
Insulin was added to protect the cardiomyocytes from
stress by enhancing myocardial metabolic recovery. How-
ever, the stimulation of glycolysis during ischemia may be
detrimental because of an accumulation of metabolic end
products. Because insulin stimulates pyruvate dehydroge-
nase activity, the addition of sodium pyruvate in the
medium may help in directing glycolysis and preventing
accumulation of metabolic end products like lactate, thus
resulting in improved aerobic metabolism (Rao et al. 1998;
Duarte et al. 2006). Horse serum along with nonessential
49
amino acids and glutamine reduced the overgrowth of
nonmyocyte cell-like fibroblasts (Fioramonti et al. 1955),
when compared to the maintenance medium containing
M199, which was used by earlier workers (Simpson and
Savion 1982; Song et al. 2000; Nickson et al. 2007). It has
already been known that α2-macroglobulins from horse
serum in combination with seromucoids from fetal calf
serum promote rapid growth of embryonic mouse cells
(Healy and Parker 1966). Although the fetal calf serum-
containing medium was used primarily for inactivation of
trypsin, it has an added advantage that it also contains
several growth factors besides differentiation factors needed
for the growth and survival of cells (Bryja et al. 2006).
Cells can be plated on dishes pretreated with collagen,
fibronectin, laminin, and gelatin or by using Primaria
(Falcon Plastics) or Pronectin (Promega, Madison, WI)-
coated culture plates (Chlopclkova et al. 2001). It has been
reported that laminin pretreatment was a less suitable
matrix for the neonatal murine cardiomyocyte cultures,
while cardiogel remains the most suitable matrix (Bick
et al. 1998). In our study and previous reports also (Song et
al. 2000; Matsuura et al. 2004), 1% gelatin has been used to
coat culture dishes, as fibroblasts easily adheres to a
collagenous extracellular matrix like gelatin (Haas et al.
1984). Thus, by using gelatin and in the absence of growth
supplements as well as nonmyocyte growth inhibitors, we
have developed a protocol for culturing neonatal mice
cardiomyocytes for a longer period of time, which is
efficient as well as cost efficient. This modified protocol
for culture of neonatal cardiomyocyte may help to study the
various stress conditions such as hypoxia.
Acknowledgments 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).
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