Download 5.2 Culture of Neonatal Cardiomyocytes

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Culture of Neonatal Cardiomyocytes
Aida Salameh and Stefan Dhein
Introduction
In many experimental approaches in cardiovascular research it is necessary to study
processes in cultured cells. There are two options at present, either adult cardiomyocytes (preferably rat) are studied (see chapter on culture of adult cardiomyocytes
by K-D. Schlüter and H.M. Piper) or neonatal rat cardiomyocytes are investigated. The
latter method has the advantage that the cells grow and divide (at least several times)
provided the neonatal rat was not older than 48 h. These cells form spontaneously
beating clusters within about 2–3 days.
The technique of isolating and culturing spontaneously beating cells from embryonic, foetal or newborn heart tissue started in 1912 using embryonic chick hearts
(Burrows 1912). At the same time the first reports on sub-cultivation of cells appeared
(Carrel and Burrows 1910) and the technique of tryptic digestion of tissue was described (Rous and Jones 1916). Despite these early successful attempts, it was the
1950ies before these techniques were used by a broader scientific community. It
started with the description of embryonic chick heart cell cultures by Moscona in
1952 and Cavanough (1955). The first neonatal rat heart cell culture was then established by Harary and Farley (1960). It was shown in the early studies that these cells
started to beat spontaneously and synchronized when they came into contact, forming synchronously beating clusters. Moreover, it was observed that these cells generated typical cardiac action potentials and might therefore form an interesting model
for electrophysiologists (Fange et al. 1956). Furthermore, it was shown in the 1960s
and 1970s that these cells responded to pharmacological and physiological stimuli
similarly to the adult heart (using e.g. isoprenaline) so that they seemed to be suitable
as a model system for pharmacologists. For a more complete review of species and
culture techniques for heart cells see Pinson et al. (1987).
However, when comparing results obtained from these cultures caution is necessary and the methods of isolation, cultivation and coating and the age of the cells have
to be taken into account. These cells change with time and may up- or down-regulate
the expression of certain proteins depending on the status of the culture, e.g. depending as to whether the cells are more or less isolated or are in close contact.
While adult cells are post-mitotic and do not divide, embryonic heart cells may
divide. Cells obtained from newborn rats are somewhat in between and can divide
several times until they become post-mitotic, which resembles the normal behaviour
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in the intact animal with the heart cells becoming post-mitotic on day 21 after birth
(Pinson 1990). However, the cardiomyocytes’ mitotic cycle seems to be 2.5-fold longer
than that of non-cardiomyocytes (Kasten 1972). The cardiomyoblast can synthesize
myosin and divide, thus representing a cell between an embryonic cell and a postmitotic adult cardiomyocyte. In the neonatal rat heart, cardiomyocytes or cardiomyoblasts of various stages of development are present. On day 1 after birth it has been
estimated that about 55% of the cells are dividing cardiomyoblasts whereas on day 4
this number drops down to 40% (Masse and Harary 1981). This is important to notice,
since it shows that the type of culture depends critically on the age of the newborn
rats and should be exactly documented. In order to obtain comparable cultures, one
should use animals of the same post-birth day for all cultures. This may also affect the
yield of certain cell types and the resistivity of the cells to the proteolytic treatment.
Cultures of neonatal rat cardiomyocytes are widely used today for patch clamp
experiments, for experiments on propagation of action potentials using optical dyes
(Rohr 1995) (and see chapter 3.1) and for investigation of regulation of protein synthesis using Western blots (see chapter on Western blots by A. Salameh) or PCR. Moreover, these cultures may be used for histological or immunohistological experiments.
Description of Methods and Practical Approach
For cultivation of neonatal rat cardiomyocytes neonatal rats of 12–48 h are used. If the
rats are older than 48 h, in our experience the cells do not grow. In most protocols, the
rats are killed by decapitation and the heart is removed and transferred to a phosphate
buffer on ice. Thereafter, the associated tissue and the atria are removed (optionally
the atria may be used to cultivate atrial cardiomyocytes), and the hearts are minced
with scissors into small pieces of about 1 mm length. Next, the tissue has to be digested using collagenase and protease as digesting enzymes. This step is carried out
at 37 °C to allow the enzyme to be active. After 5–10 min of digestion the supernatant
is collected and the digestion is stopped with FCS/medium. The remnant pellet is
again subjected to the digesting enzyme solution for another 5–10 min period. These
steps are repeated several times, until all the material is digested and finally stopped
with FCS/medium.
Subsequently, the cell-containing solution is centrifuged and the pellet resuspended. Next, it is necessary to remove non-cardiomyocytes, which can be achieved using
a differential attachment technique, i.e. cells are incubated for 1 h at 37 °C in a cell
culture flask so that the non-cardiomyocytes such as fibroblasts or endothelial cells,
which attach more easily to plastic surfaces than cardiomyocytes, can attach to the
surface. After 1 h the supernatant is collected and plated in Petri dishes coated with
either gelatin or laminin (which is however rather expensive) for cultivation. For the
first day we use a medium containing 5% foetal calf serum (FCS). On the second day
and for the further cultivation FCS is reduced to 1%. Cell culture medium should be
changed every day or at least every second day. In order to avoid excessive growth of
fibroblasts, 10% horse serum should be added. Using this technique, clusters of spontaneously beating cardiomyocytes are obtained and can be observed after the second
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day. If the cells are used for patch clamp experiments they are best at days 3 to 5.
Thereafter, the cells become flat and are difficult to patch. Moreover, in our experience
older cells are rather fragile and do not tolerate a patch pipette well. Within 4 to 7 days
the cultures become more or less confluent (depending on the number of cells which
were initially seeded) and may be used for investigation of protein synthesis or biochemical experiments.
Many variations of this protocol are possible using other enzymes or mixtures,
times, and temperatures. Below, we give an example as it is routinely used with good
success in our laboratory. The optimal enzyme depends on the age and species of the
animal. Often crude enzyme preparations are found to be more efficient than purified
enzymes. On the other hand, the enzyme mixture may alter the cardiomyocytes or
cardiomyoblasts as well. Thus, it is recorded that trypsin can damage myocardial cells.
If purified collagenase is applied, this has to be combined with trypsin or another
protease, since collagenase alone is a poor digesting enzyme. Enzymes often used are
pronase and elastase (e.g. for embryonic chick heart cell cultures) or crude trypsin
preparations, which also contain chymotrypsin and elastase. When using crude
trypsin, one should document the activities of chymotrypsin and elastase (which may
vary from batch to batch, the manufacturers can normally provide information on the
other enzyme activities), in order to allow the finding of comparable batches of the
enzyme if the old batch is running out. An alternative may be the use of Viokase
(Viobin Corp.), a mixture of proteolytic enzymes, which is more consistent in its composition than crude preparations. It is also possible to purify an enzyme mixture from
a crude preparation. Thus, Speicher and McCarl (1978, 1978a) described a procedure
for purifying a mixture of chymotrypsin, elastase and trypsin from a crude trypsin
preparation, resulting in a preparation which was highly efficient but less toxic to the
cells than the crude preparation.
Although some experimenters argued that trypsin may lead to damage of the cardiomyocytes, trypsin is widely and successfully used in cell isolation protocols, mostly
in concentrations of 0.05 to 0.1%. To keep cell damage to a minimum trypsinization
cycles should be kept short, i.e. 10 to 20 min. Moreover, one may add glucose during
the dissociation, which has been shown to result in better cell yield. Another factor
which has to be controlled is the purity of water used. In our experience the quality
of water used is very important.
Since growth and differentiation of cells is normally controlled by cell contacts,
the cell types involved (i.e. the non-myocardial cells), humoral factors, the extracellular matrix and hormones, the extracellular milieu of the cells in culture, the medium,
and the frequency of changes of the medium are highly important. Media used in
neonatal rat heart cell culture are Ham’s F10, M199 (which can both be obtained from
most manufacturers) and CMRL 1415ATM (Connaught Medical Research Laboratories). In our lab we normally use M199. Since most of these media contain NaHCO3,
they require constant CO2 presence (around 5%) in the incubator, to maintain a pH of
7.4. The exact concentration of CO2 has to be determined. Most manufacturers give
information on their media.
In most protocols, sera are added, such as foetal calf serum (FCS), foetal rat serum,
horse serum or embryo extracts. These sera have detoxifying properties, help the cells
to attach and promote cell growth by the presence of a number of hormones and
Culture of Neonatal Cardiomyocytes
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growth factors. The composition of a serum may change from batch to batch, which
makes it necessary to test a batch of serum first and, if it works well, buy a lot of it.
Unfortunately, the use of serum promotes the growth of non-myocardial cells, which
will, due to their shorter cell cycle, overgrow the cardiomyocytes. Thus, the amount of
serum may also be critical. Most groups use FCS in concentrations of 1 to 10% as an
additive to their media. A compromise, with which we have had good experiences,
may be to use a higher FCS concentration (5%) on the first day after isolation, and to
reduce the serum content to 1% on the second day. Horse serum (about 10%) often is
added since it is said to reduce fibroblast growth.
Alternatively, serum-free cultures may be used. This will reduce the number of
non-myocyte cells (Kessler-Icekson 1987; Claycomb 1980). In that case it is important
to coat the Petri dishes with fibronectin or collagen to allow cell adhesion. Some
authors coat their dishes with FCS first, then wash the dish and afterwards use it for
serum-free culture. The FCS serves as an attachment factor in these protocols. In
serum-free cultures, nutrients, hormones and fatty acids may be supplemented. In
such protocols, the serum glucoprotein fetuin has been shown to be an essential factor to be added (Claycomb 1980; Mohamed et al. 1983).
Glucocorticoids, which have been shown to maintain the beating capacity of the
cardiomyocytes and to lead to growth retardation of non-cardiomyocytes, may be
added Ascorbic acid and selenium are often used as antioxidants in serum-free cultures. Transferrin may be used for detoxification of toxic metals and as a carrier for
iron. Albumin may be used in order to carry fatty acids. Other hormones such as insulin, adrenaline or noradrenaline may be used depending on the requirements of the
experiment. Hormonally defined media may also be used (Kessler-Icekson et al. 1983).
The use of growth factors in serum-free cultures is questionable since this will enhance non-cardiomyocyte growth (which should be avoided by using the serum-free
protocol). However, serum-free protocols may also be useful for the investigation of
hormone or drug/cardiomyocyte interactions in order to exclude interference with
serum factors.
For cell culture either glass Petri dishes, plastic Petri dishes or plastic culture flasks
may be used. They should be coated with either gelatin, FCS, fibronectin, laminin or
collagen. Gelatin is an easy to use, mostly collagen-containing coating factor which is
used in many labs. If glass is used, the quality of glass is important, since the polarity
of the glass surface may be different between various glass qualities. If cells are to be
seeded in the culture dishes, it is necessary to determine the cell number/ml using e.g.
a Neubauer counting chamber. If cells are used for patch clamp experiments the cell
number should be low. Otherwise, if cells are used for biochemical experiments, it is
often desired to have a more or less pure cardiomyocyte culture with a minimum
of non-myocardial cells. This is achieved by seeding higher cell numbers such as
106 cells/ml which will lead to confluence within 24–48 h. Reaching confluence means
contact inhibition to non-cardiomyocytes and thus prevents overgrowth of the culture with these cells.
While the heart contains about 50% non-cardiomyocytes, mostly fibroblasts/
fibrocytes and endothelial cells, a mixture somewhat enriched in cardiomyocytes is
obtained after the cell isolation protocol with about 80% cardiomyocytes and 20%
non-cardiomyocytes. It has been assumed that a minimum of non-cardiomyocytes is
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necessary in vivo and in vitro for proper functioning of the cardiomyocytes. On the
other hand these non-cardiomyocytes sometimes complicate the interpretation of the
results.
Within the finally obtained cell culture the cardiomyocytes can be identified by
their beating capacity, by their action potentials or visually by their dense cytoplasm,
well-developed mitochondria and Golgi system, myofibrils, intercalated disks and
cross-striated contractile proteins. Moreover, they express cardiotypic proteins such
as α-actinin and troponin I.
From our point of view the most important rule in cell culture to obtain comparable results is a high standardization of protocols, solutions, enzyme preparations,
water, media and sera.
In-Vitro Techniques
Example
The following equipment will be needed: a sterile hood, best equipped with a UV-sterilized area, an inverted-phase microscope, an incubator (37 °C, 5% CO2), an autoclave,
a thermocontrolled water bath, a centrifuge, plastic Petri dishes, forceps, scissors, scalpel, 10 ml pipettes, 8 Falcon tubes (50 ml), 10 ml syringes. Scissors and forceps need
to be sterile.
The following protocol is our daily use protocol. It should be stated that this is one
possible example. Other protocols or modifications may also work. The interested
reader may adapt this protocol to his needs according to the outlines given in the
paragraphs above.
Prepare ice in a styropor container and the following solutions:
1. stop-solution
 M199 (Hanks salts, HEPES): 474.5 ml
 5% FCS: 25 ml
 PenStrep: 0.5 ml
2. collagenase solution
 PBS-glucose: 50 ml
 Bovine serum albumin (BSA): 0.5 g
 Collagenase Type II: 50 mg (204 U/mg) (total about 10.000 U)
3. day 1 medium (500 ml)
 M199 (Hanks salts, HEPES): 424,5 ml
 5% FCS: 25 ml
 10% horse serum: 50 ml
 PenStrep: 0.5 ml
4. phosphate buffered saline (PBS) with glucose (50 ml)
 NaCl 137 mM: 401 mg
 KCl 2.7 mM: 0.01 g
 Na2HPO4 8.3 mM: 0.073 g
 KH2PO4 1.5 mM: 10.2 mg
 Glucose 20 mM: 0.198 g
 pH 7.4
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5. penicillin/streptomycin solution (PenStrep)
 100 mg/ml penicillin
 100 mg/ml streptomycin
6. gelatin 1% (for coating of Petri dishes)
 1.5 g gelatin in 150 ml H2O; then autoclave
7. Dulbecco’s wash solution
 NaCl 137 mM
 KCl 2.68 mM
 Na2HPO4 6.48 mM
 KH2PO4 1.47 mM
 MgCl2 0.49 mM
 MgSO4 0.81 mM
 CaCl2 0.9 mM
 pH 7.2
8. day 2 medium (500 ml)
 M199 (Hanks salts, HEPES): 444.5 ml
 1% FCS: 5 ml
 10% horse serum: 50 ml
 PenStrep: 0.5 ml
All solutions must be sterile either by sterile filtration or autoclaving.
Place the following items under the sterile hood: PBS-glucose in a large Petri dish,
a glass with 70 % ethanol, a sterile filter and a syringe, collagenase solution, stop solution, several sterile 50 ml Falcon tubes. First decapitate the neonatal rats (10 for 1 cultivation) using large scissors, open the thorax, and remove the hearts with small scissors and forceps. The hearts have to be transferred immediately to a Petri dish with
10 ml ice-cold PBS-glucose to avoid ischemia. Remove the blood by gently shaking.
Transfer the dish under the sterile hood. Warm the stop solution and the medium (day
1) in a water bath at 37 °C. Fill a second Petri dish with ice-cold PBS-glucose solution.
Now remove the supernatant solution but leave about 1 third of the solution to
avoid drying of the tissue. Next, the atria and the associated tissue have to be removed
using small scissors and forceps (instead, it might be interesting to use only the atria).
The ventricles are transferred to the second dish with 10 ml ice-cold PBS-glucose. The
ventricles are now minced into small pieces of about 1 mm length and then the total
10 ml containing the hearts are transferred into a sterile Falcon tube (50 ml). 7 ml of
the collagenase solution are added and the Falcon tube is closed. Then, gently shake
the solution in a 37 °C water bath for 5–6 min. Thereafter, the supernatant, which
contains the first isolated cells, is transferred into another sterile 50 ml Falcon tube
(FT-II) and 7 ml stop solution are added. With the remaining tissue the same procedure is repeated, i.e. 7 ml collagenase solution are added again, the tube is shaken for
another 5–6 minutes at 37 °C. Then, the supernatant is transferred into the Falcon
tube (FT-II) and 7 ml stop solution are added. This procedure is repeated until the
50 ml of collagenase solution are completely used up.
Next, the solution in the Falcon tubes containing the cardiomyocytes is centrifuged at 700 rpm/min for 5 min. The supernatant is removed and the resulting cell
pellet is resuspended in 13 ml day 1 medium. Thereafter, the solution has to be trans-
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Figure 1
Fluorescence microscopy of ventricular neonatal rat cardiomycytes with anti-α-actinin staining. For
coloured version see appendix
ferred to a 75 cm2 culture flask and incubated in the incubator at 37 °C (5% CO2) for
1 hour. During that time, mainly non-cardiomyocytes attach to the plastic surface.
During this hour the Petri dishes for the cardiomyocyte culture are coated with 1%
gelatin solution. After 1 h the supernatant of the culture flask is removed and transferred to a Falcon tube for cell counting. Thereafter, the cell suspension is diluted with
day 1 medium to the correct cell number (e.g. 1 million/ml) transferred to the Petri
dishes and incubated for 24 h.
Next day, the Petri dishes are washed twice with Dulbecco’s wash solution. Then
the cells are fed with the day 2 medium Subsequently, the medium should be changed
every 24–48 h until the cells are used for the experiments. Normally, you should observe the first spontaneously beating cells or cell clusters on day 2.
The culture should be monitored by histological /immunohistological methods.
The cells should be stained for α-actinin, actin, troponin I and connexin 43. The contractile proteins should exhibit a cross-striated pattern. Cx43 should be localized at
the cell borders and at the cell-cell contact zones. Figure 1 shows neonatal rat
cardiomyocytes.
Troubleshooting
The most common problems with this type of primary cell culture are infections. In
case of an infection, the infected culture dishes have to be removed. To avoid infections, sterility of all solutions has to be controlled very strictly. Thus, the gelatin solu-
Culture of Neonatal Cardiomyocytes
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tion may be infected, especially if it is used for a long time, or the FCS solution by
using a pipette several times. In order to find the source of infection, it is sometimes
helpful to incubate the suspected solutions for 24 h in an incubator at 37 °C. The use
of additional antibiotics/antimycotics etc. should be strictly avoided since you may
select multiresistant bacteria this way. To keep infections at a minimum, you may wash
the neonatal rats with isopropanol prior to opening the thorax.
Another common problem is a small cell yield after the isolation steps. This may
be related to ischemia during the isolation procedure. It is essential to perform the
first steps on ice and to avoid all unnecessary delays. Another factor, which might
compromise the result, is the shaking of the collagenase/cell solution. It seems necessary to shake this solution gently and not to stir it. It may be suspected that during this
step the cells are more susceptible to mechanical stress.
Sometimes, after a good cell yield at the first day, the cell number is markedly reduced after the second day. A typical problem is the washing step to remove the day
1 medium. It is essential to use the Dulbecco’s wash solution as stated in this chapter,
i.e. containing Ca++ and Mg++. Some Dulbecco’s wash solutions which can be obtained
commercially do not contain calcium. If these, i.e. Ca++-free solutions, are used for
washing, the cardiomyocytes will lose their contact to the surface, since many of the
contacts are calcium-dependent.
Another problem often encountered with neonatal rat heart cell culture is contamination with non-cardiomyocytes. There are several strategies discussed in the
first part of this chapter to keep the number of contaminating cells at a minimum.
However, it should be noted that a number of authors have argued that a minimum
number of non-cardiomyocytes (10–20%) may be necessary to maintain a proper
functioning cardiomyocyte culture, as in the heart there are also non-cardiomyocytes,
which may influence the cardiac cells by humoral and non-humoral factors such as
direct contact. In order to minimize the percentage of non-cardiomyocytes, differential attachment as described above may be used. It might be useful to adapt the times
to special requirements and to monitor the two fractions (that in the culture flask and
that in the supernatant). In addition, the use of 5–10% horse serum is recommended
to retard fibroblast growth. Since non-cardiomyocytes are stimulated with FCS, we
recommend the reduction of FCS at the second day to 1% (day 1 medium 5%; day 2
medium 1%). This might be adapted. Another possibility is the use of a serum-free
culture method (see above); Moreover, several investigators recommend the addition
of 7-β-OH cholesterol. Finally, since the non-cardiomyocytes exhibit a faster cell cycle
with higher DNA synthesis, DNA synthesis may be inhibited by BrdU incubation and
subsequent UV irradiation or by other chemical inhibitors of DNA synthesis. However, it is questionable whether this may not also alter the physiology of the cell culture and might compromise the results. If such treatments are used, very careful
controls have to be performed to exclude effects of the treatment.
References
Burrows MT (1912) Rhythmical activity of isolated heart muscle cells in vitro. Science 3:90-92
Carrel A, Burrows MT (1910) Culture de tissus adultes en dehors de l’organisme. CR Soc Biol (Paris) 69: 293-294
Cavanough MW: Pulsation migration and division in dissociated chick heart cells. J Exp Zool 128:573-589, 1955
Claycomb WC (1980) Culture of cardiac muscle cells in serum-free medium. Exp Cell Res 131:231-236
Fange R, Persson H, Hesleff T (1956) Electrophysiologic and pharmacologic observations on trypsin disintegrated
embryonic chick hearts cultured in vitro. Acta Physiol Scand 38:173-183
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Harary I, Farley B (1960) In vitro studies of isolated beating heart cells. Science 131:1674-1675
Kasten FH (1972) Rat myocardial cells in vitro: mitosis and differentiated properties. In Vitro 8:128-150
Kessler-Icekson G, Wassermann L, Yoles E, Aampson SR (1983) Characterization of cardiomyocytes cultured in serumfree medium. In: Fischer G, Weiser RJ (eds) Hormonally defined media. A tool in cell biology. Springer Verlag,
Berlin, p. 383
Kessler-Icekson G (1987) Cardiomyocytes grown in serum-free medium. In: Pinson A (ed) The heart cell in culture.
CRC, Boca Raton, p 23-28
Masse MJO, Harary I (1981) The use of fluorescent antimyosin and DNA labelling in the estimation of the myoblast
and myocyte population of primary rat heart cell cultures. J Cell Physiol 106:165-172
Mohamed SNW, Holmes R, Hartzell CR (1983) A serum-free chemically defined medium for function and growth of
primary neonatal rat heart cell cultures. In Vitro 19:471-478
Pinson A, Padieu P, Harary I (1987) Techniques for culturing heart cells. In: Pinson A (ed.) The heart cell in culture,
vol 1. CRC, Boca Raton, p 7-22
Pinson A (1990) Neonatal rat heart muscle cells. In: Piper HM (ed.) Cell culture techniques in heart and vessel research.
Springer Verlag, Berlin, p 20-35
Rohr S (1995) Determination of impulse conduction characteristics at a microscopic scale in patterned growth heart
cell cultures using multiple site optical recording of transmembrane voltage. J Cardiovasc Electrophysiol 6:
551-568
Rous P, Jones FS (1916) A method for obtaining suspensions of living cells from fixed tissues and for plating individual
cells. J Exp Med 23:549-555
Speicher DW, McCarl RL (1978) Isolation and characterization of the proteolytic enzyme component from commercially available crude trypsin. Anal Biochem 84:205-217
Speicher DW, McCarl RL (1978) Evaluation of a proteolytic enzyme mixture isolated from crude trypsins in tissue
disaggregation. In Vitro 14:849-853