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Protocol for Culture of Cardiomyocyte

Protocol for Culture of Cardiomyocyte

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Published by Prasath
Primary culture of cardiomyocytes has been widely used as a valuable tool for pharmacological and
toxicological studies. However, the fact that heart is a solid organ and cardiomyocytes do not proliferate
after birth makes the primary myocardial culture a tedious job. The present study reports an improved
method for rapid isolation of cardiomyocytes, as well as the culture maintenance and quality assurance.
Primary culture of cardiomyocytes has been widely used as a valuable tool for pharmacological and
toxicological studies. However, the fact that heart is a solid organ and cardiomyocytes do not proliferate
after birth makes the primary myocardial culture a tedious job. The present study reports an improved
method for rapid isolation of cardiomyocytes, as well as the culture maintenance and quality assurance.

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Published by: Prasath on May 22, 2009
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02/02/2013

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-1
An optimized protocol for culture of cardiomyocyte from neonatal rat
Jiajia Fu, Jie Gao, Rongbiao Pi and Peiqing Liu*
Department of Pharmacology and Toxicology, Sun Yat-sen University, Zhongshan 2 Road, Guangzhou,510080, China; *Author for correspondence (e-mail: sps02@gzsums.edu.cn; phone: +8620-87334613; fax:+8620-87334718)
Received 16 October 2005; accepted in revised form 3 January 2006
Key words:
Cardiomyocytes, Culture, Neonatal, Precondition, Protocol
Abstract
Primary culture of cardiomyocytes has been widely used as a valuable tool for pharmacological andtoxicological studies. However, the fact that heart is a solid organ and cardiomyocytes do not proliferateafter birth makes the primary myocardial culture a tedious job. The present study reports an improvedmethod for rapid isolation of cardiomyocytes, as well as the culture maintenance and quality assurance.The whole culture process can be shortened to 3.5 h by reducing enzyme digestion period. Moreover, thenew protocol guarantees cell yield and viability, and produces more than 95% cardiomyocytes in culture.The cardiomyocytes can respond to Angiotension II stimulation with increased protein synthesis, sug-gesting the practical value of this new culture method.
Introduction
Since Harary and Farley first separated Wistarneonatal rat cardiomyocytes and succeeded inmaking the myocardiocytes exhibit spontaneouslybeating activity for 40 days
in vitro
(DIV) (Harryand Farley 1960), primary culture of cardiomyo-cyte has been widely applied in the basic cardio-logical research. One advantage in doingexperiments with neonatal rat cardiomyocytes islack of the influences of hemodynamic factorsexisting
in vivo
. In addition, in cell culture it isfeasible to control other concomitant factorsartificially.The culture method of neonatal rat cardio-myocytes established by Harary and Farley hasbeen modified by many scientists in the past40 years (Harary and Farley 1963). Two isolationsteps are essential for a successful culture. One isthe enzyme digestion step for dissociating cellsfrom heart tissue. The other is the purification stepfor eliminating non-muscle cells. The latter iscritical for ensuring a constant proportion of myocytes. Several techniques have been applied toreach this goal, including differential attachmenttechnique (Blondel et al. 1971), deprivation of serum (Shields et al. 1988), density gradient cen-trifugation (Flanders et al. 1995; Harada et al.1998), and using chemical reagents to inhibit non-muscle cells proliferation (Simpson and Savion1982). To make a good culture with high myocytesyield, repetitive trypsinization of heart tissue inshort periods is often recommended, which gen-erates a higher proportion of undamaged musclecells, other than a single digestion for longer time(Mark and Strasser 1966). Normally, the incuba-tion time with trypsin depends on the amount of undigested tissue. Although repetitive digestioncan give good yield, it could also lead to poor cellviability when the minced tissue is exposed to
Cytotechnology (2005) 49:109–116
Ó
Springer 2006DOI 10.1007/s10616-006-6334-6
 
enzyme solution for too long. That is because theheart is one of the most delicate organs sensitive toenvironmental changes. Therefore, the isolationand culture of neonatal cardiomyocytes requireexperience and skills for ensuring good yield andhigh quality cell culture. Moreover, because car-diomyocytes lose their ability to proliferate shortlyafter birth, growth of heart tissue is governed bycell growth rather than by proliferation. This fea-ture of cardiomyocytes does not allow cell propa-gation and requires repetitive primary cultures formultiple experiments. Hence, the application of cardiomyocyte culture is somehow limited becauseof the heavy preparation duty.The present study reports a convenient, reliableand time-saving protocol for making consistentcultures of high-yield and high-quality cardio-myocytes. By decreasing the digestion periods, thewhole culture process only takes 3.5 h, including a1.5-h purification step. The yield is 3
·
10
6
cellsfrom one animal with 90% viable cells, which iscomparable to the conventional culture protocol(Paul Simpson 1985)
Materials
Equipments
Magnetic stirrer (with heating function) (IKA
1
,RH-T/C)Liquid scintillation counter (Beckman
2
, LS 6000)
Solutions/supplies
Culture medium: One packet of DMEM powder(GIBCO
3
, 12800-017) is dissolved in 1000 ml of distilled water, 2.2 g sodium bicarbonate and25 mM Hydroxyethyl piperazine ethanesulfonicacid (HEPES) (Sigma
4
, H4034) are added andadjust the pH value to 7.3. This solution is steril-ized by filter-sterilization. The following sterilesolutions are added to complete the DMEM: 10%(v/v) heat-inactivated fetal bovine serum (HangZhou Sijiqing
5
, 050416), Antibiotics stock mixture(Hyclone
6
, SV30010).Enzyme solution: 0.08% trypsin solution isfreshly prepared to ensure the enzyme activity.Dissolve trypsin (Sigma
4
, T8003) in Ca
2+
andMg
2+
-free PBS buffer either at room temperaturefor 4 h with agitation or at 4
°
C overnight. Ster-ilize the solution by filtration through a 0.22
l
mfilter.PBS solution: one packet of Ca
2+
and Mg
2+
-free PBS powder (Boster
7
, AR0030) is dissolved in2 l dH
2
O. The pH value is adjusted to 7.3. Thesolution is sterilized either by autoclaving or filter-sterilization.Deoxyribonuclease I (sigma
4
, 31135) is dissolvedin sterile PBS buffer to make a stock solution of 5 mg ml
À
1
, dilute the solution to 50
l
g ml
À
1
withtrypsin solution before use.5-Bromo-2
¢
-deoxyuridine (BrdU) (Aldrich
4
,858811) is dissolved in sterile dH
2
O to a stocksolution of 100 mmol l
À
1
, final concentration is0.1 mmol l
À
1
.Angiotensin II (Sigma
4
, A9525) is dissolved insterile dH
2
O to a stock solution of 10
À
3
mol l
À
1
.Dilute it to appropriate concentration with serumfree medium before use.Cell culture plates (diameter 35 mm) and flasks(Greiner bio-one
8
, 657 160, 690 170 respectively).Anti-
a
-sarcomeric actin ((Sigma
4
, A2172) isdiluted to 1:600 with dH
2
O before use.SABC immunoenzymatic staining kit (Boster
7
,AR0030)
Procedures
Preparation of cardiomyocyte culture
1. Anesthetization and sterilization: Rat pups(Sprague-Dawley or Wistar rats) at the age of postnatal day 1–3 were sacrificed by ethyl ether.The animals were decontaminated with 75% eth-anol, and transferred to a Luminer flow hood.
1
IKA
Ò
Works Guangzhou, China
2
Global Medical Instrumentation, Inc 6511 Bunker LakeBoulevard Ramsey, Minnesota, USA
3
GIBCO, 1600 Faraday Avenue Carlsbad, California 92008
4
Sigma Chemical Corp., St. Louis, MO, USA
5
Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.Hangzhou, China
6
HyClone, Logan, UT, USA
7
Boster Biological Technology Ltd, Wuhan, China
8
Greiner bio-one International AG., Bad Haller Strasse 32 A-4550 Kremsmuenster
110
 
2. Dissection (performed on ice): Surgically re-move the beating heart from animals immediately,and keep it in cold Ca
2+
and Mg
2+
-free PBSbuffer. Ventricles were excised and transferred tofresh ice-cold PBS buffer and were minced with finescissors into 1–3 mm
3
pieces after washing bloodaway from the heart lumen. Red blood cells wereremoved by instant centrifugation for two times.3. Preconditioning (20 min): A preconditioningstep was introduced in the present protocol priorto trypsinization. The minced tissue was trans-ferred to a 40 ml conical flask containing trypsinsolution (0.08%, 0.5 ml per rat) and a smallmagnetic bead. The flask was then settled on icefor 20 min, and shaken every 3 min for bettermixing.4. Trypsinization (10 min): Following precon-ditioning, the tissue was digested in the conicalflask at 37
°
C for 10 min, which was subjected toconstant stirring (150–200 rpm).5. Centrifugation (5 min): After trypsinization,cells were dispersed from the tissue by gently pi-peting. The cell suspension was settled on ice forseveral seconds. The supernatant was carefullytransferred to a 15 ml centrifuge tube. Trypsinactivity was inhibited by adding a mixture of trypsin inhibitor and cold culture medium sup-plemented with 10% FBS (1:1, v/v). Cell pellet wasformed by spinning at 1000 rpm for 5 min, andwas resuspended in 2 ml warm culture medium.6. Repeating trypsinization: The remainingtissue in the conical ask from step 5 wascontinuously digested by adding 5–10 ml freshpre-warmed trypsin solution containing DNase I(0.05 mg ml
À
1
). Depending on the amount of undigested tissue, trypsinization and centrifuga-tion steps were repeated for 2 to 3 times(25–35 min).7. Cell harvest (10 min): Cell suspension fromall dissociated steps was pooled in one centrifugetube and settled for 5 min. The suspension wasgently transferred to a new centrifuge tubeexcluding the precipitates on the bottom. Cellswere harvested by centrifugation for 5 min at1000 rpm. Finally the cells were plated in a 40 mltissue culture flask and incubated at 37
°
C in ahumidified atmosphere (5% CO
2
, 95% air).8. Purification (1.5 h): Myocardiocytes enrichedculture is obtained through the following twosteps. Since non-myocardiocytes attach to thesubstrata more readily than myocardiocytes,firstly, cells harvested from step 7 were incubatedfor 1.5 h to allow the attachment of non-myo-cardiocytes. The majority of myocardiocytesremained in culture medium. The suspended cellswere collected and plated at a density o2
·
10
5
ml
À
1
into a new tissue culture flask. BrdU(0.1 mmol l
À
1
) was then added to the culturemedium for 48 h to prevent proliferation of non-myocardiocytes that might be present in the cul-ture.9. Cultivation: Generally, cells isolated from 2 to3 hearts can be seeded in one 40 ml culture flask.The cells should not be disturbed during the initial24 h. The culture medium was replaced with freshmedia without BrdU for every 48 h.
Cell yield and viability evaluation
The yield and viability of the culture was moni-tored by dye exclusion using trypan blue (0.4%).Mix 1 drop of trypan blue (0.4%) with 9 drops of cell suspension and allow 1–2 min for absorption.Cells excluding the staining are considered viableand the percentage of non-blue cells is used as anindex of viability. Count both the total number of cells and the number of stained (dark) cells by ahemocytometer for measuring the yield and via-bility as follows:Yield
¼ð
total number of cells in four grids
=
4
ÞÂ
10
4
Âð
cell suspension volume
Þ
Viability
ð
%
Þ¼ð
Total cells counted
À
stained cells
Þ
=
total cells counted
Â
100
Immunoenzymatic staining assay
Since
a
-sarcomeric actin is considered as a specificprotein in cardiomyocytes, a mouse monoclonalanti-
a
-sarcomeric actin (1:600, Sigma) wasapplied as the primary antibody to identify car-diomyocytes in the culture. A goat anti-mousebiotinylated immunoglobulin conjugated withavidin-biotinylated horseradish peroxidase wasused as the secondary antibody followed by ABC111

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