Supplemental Material

A new protocol for isolating cardiac myocytes used in patch-clamp studies

Overview

We describe here a new protocol for isolating rat atrial cells that minimizes variations

associated with the conventional Langendorff perfusion procedure (1), eliminates the

collagenase-screening process, and consistently yields viable single cells. Instead of perfusion

with collagenase at 37˚C, the primary digestion is carried out by perfusion with papain at room

temperature. Collagenase digestion is subsequently performed in a 32˚C water bath to disperse

single cells. Similar cell isolation results can be consistently obtained with papain and

collagenase from different vendors. When the cells are to be used for electrophysiological

studies, a final digestion step is added to facilitate the formation of Giga-ohm seals. This

protocol is slightly modified for isolating mouse atrial and rat and mouse ventricular cells (see

the end of this report for details of the modification).

Cell isolation

Male Sprague Dawley rats (150-350 g) were anaesthetized using pentobarbital (i.p. 80

mg/kg). Heparin (dissolved in 0.9% NaCl solution at a concentration of 500 units per milliliter,

filtered with a 0.2 m syringe filter, and stored at –20C until use) was then injected into the tail

vein (1 unit per gram body-weight) to prevent blood clotting. Three minutes after the heparin

injection, an incision was made across the abdomen below the diaphragm and the chest was

opened to fully expose the heart and lungs. The heart and lungs were cut from the body using

scissors by cutting close to the spine to preserve the descending aorta. The heart, together with

other extraneous tissues, was rapidly transferred into an ice-chilled 20 ml beaker containing 10
ml BDM-HEPES solution (see below for a list of the solutions used) supplemented with 10

unit/ml heparin (BDM: 2,3-butanedione monoxime). The heart was allowed to be chilled on ice

for 10 minutes to stop contraction.

The heart was then transferred into a petri-dish (with a piece of gauze pad on the bottom)

containing BDM-HEPES solution. After the lungs were removed, the aorta was carefully

separated from the surrounding tissue. The aorta was cannulated with an infusion set (19G3/4,

Terumo Corp.) modified to be used as the cannula by flattening the tip of the needle using a file.

The back of the cannula was connected to a syringe filled with the BDM-HEPES solution. Then

the needle was inserted into the left ventricle through the aorta. Three to five milliliters of BDM-

HEPES solution were injected into the left ventricle to flush blood out through the aorta. The

aorta was then tied to the needle using size 4-0 silk suture (Fine Science Tools, Inc.). Another 3-

5 ml of BDM-HEPES solution was injected into the left ventricle to wash out residual blood in

the coronary blood vessels. After the blood was flushed out, the syringe was removed and the

cannula was connected to a peristaltic pump (MasterFlex C/L, Cole-Parmer Inc.).

Digestion step-1: At a rate of 1 ml/min, 20 ml of papain solution (10-15 unit/ml papain in BDM-

HEPES solution supplemented with 50 M MgSO4 and 50 M EDTA) was perfused through the

heart at room temperature (25 ± 1 ˚C). To activate papain, 30 to 50 mg of papain were dissolved

into 1 ml of 5 mM dithiothreitol and allowed to equilibrate for at least 5 minutes. Then, an

appropriate amount of this stock solution was added into the BDM-HEPES solutions to reach the

final papain concentration used for perfusion.

Digestion step-2: After perfusion, both left and right atria were removed from the heart. The

atrial chambers were cut open and each atrium was cut into four pieces. These tissue pieces

(inside-up) were put into a petri-dish containing 5 ml of papain solution (as described above) for

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30 minutes at room temperature. Then, the atria were further cut into small strips (about 1 mm ×

2 mm) using fine scissors under a dissecting microscope. The strips were washed twice with 5

ml BDM-HEPES-Mg solution and transferred into 5 ml BDM-HEPES-Mg solution for a ten-

minute incubation at room temperature.

Digestion step-3: At 10 minutes, the BDM-HEPES-Mg solution was replaced with 5 ml

Digestion solution (containing collagenase and DNase) and the strips were incubated for 30

minutes at 32 C, with gentle agitation every 10 minutes. This procedure was carried out in a 20

ml container with a flat bottom to enhance the contact between tissue and enzyme. The tissue

strips were then transferred into a 15 ml centrifuge tube containing 3 ml Glutamate-K solution,

and cells were dispersed with trituration using a fire-polished Pasture pipette (with an opening

diameter of about 2 mm). The dispersed cells in the suspension were transferred into another

tube. The dispersion procedure was repeated 3-4 times until most of the remaining tissue fell

apart. The pooled cell suspension (total of 10-15 ml) was centrifuged at 500 rpm for 10 minutes.

The supernatant was carefully removed and the cells were re-suspended into Glutamate-K

solution for experimentation.

Digestion step-4: This final digestion step is specifically designed to prepare the cells for

electrophysiological studies. After centrifugation, the cells were re-suspended into 10 ml of

Glutamate-K solution supplemented with 3 unit/ml papain and 50 unit/ml DNase-I. Following a

30-minute period at room temperature, cells were centrifuged again at 500 rpm for 10 minutes.

The cell pellet was washed with Glutamate-K solution, centrifuged and re-suspended in

Glutamate-K solution. The cells could be stored at room temperature for use within the next 6-8

hours.

Restoration of extracellular Ca2+: A Ca2+-restoration procedure was required to carry out

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experiments with the cells in a solution containing physiological concentrations of Ca2+. When

this was required, after the final centrifugation step, cells were washed with 10-15 ml of BDM-

HEPES-Mg solution, centrifuged and re-suspended in 5 ml BDM-HEPES-Mg solution. The Ca2+

concentration was adjusted stepwise to 0.05, 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 mM by adding

aliquots of 100 mM CaCl2 solution at seven-minute intervals. Cells were then placed on ice for

use within the next 6-8 hours.

Solutions and chemicals

BDM-HEPES solution contained (in mM): 120 NaCl, 5.4 KCl, 1.2 NaH2PO4, 0.1 MgSO4,

15 NaHCO3, 5 BDM, 10 HEPES (sodium salt), 5 Taurine, and 5 Glucose (glucose was added

just before the isolation procedure). The pH was adjusted to 7.4 with HCl at room temperature.

BDM-HEPES-Mg solution was the BDM-HEPES solution supplemented with 1mM MgSO4.

Digestion solution was BDM-HEPES-Mg solution supplemented with 478 units/ml

collagenase (Type II, Worthington), 2 mg/ml BSA (Sigma-A9647) and 50 units/ml DNase-I

(Sigma-D5025). DNase-I stock solution was prepared in 0.1 mg/ml BSA as aliquots of 150 l,

each containing 750 units DNase.

Glutamate-K solution contained (in mM): 120 glutamic acid, 0.3 KH2PO4, 1 EGTA, 5

MgSO4, 20 taurine, 10 HEPES (potassium salt), and 10 ATP or 10 glucose (glucose was added

before experiments). The pH was adjusted to 7.4 with KOH at room temperature.

High-K solution contained (in mM): 126 KCl, 2 MgCl2, 1 EGTA (with 3.5 KOH), and 10.5

HEPES (potassium salt). The pH was adjusted to 7.4 with HCl at room temperature.

Whole-cell bath solution contained (in mM): 130 NaCl, 5.4 KCl, 1 MgCl2, 1 CaCl2, 10

HEPES (sodium salt). The pH was adjusted to 7.4 with 1 M HCl at room temperature.

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 Whole-cell pipette solution contained (in mM): 124 KCl, 3 MgCl2, 3 Na2ATP and 10

HEPES (potassium salt). The pH was adjusted to 7.2 at room temperature with HCl.

All of the solutions, except for the digestion solution which was prepared by adding

enzymes just before the isolation procedure, were filtered with 0.2 m membrane filters and

stored at 4 C.

Pentobarbital (Nembutal Sodium Solution, 50 mg/ml, USP) was from Abbott Laboratories

(USA). BAPTA and fluo-3 were from Molecular Probes, INC. All other chemicals were

purchased from Sigma unless otherwise specified.

Additional details

More than one hundred thousand atrial cells were obtained after triturating the tissue strips

in the Glutamate-K solution. For ten batches of cells when we calculated the yield, the number

of healthy cells was 3.0 ± 1.2×105 and the number of damaged cells was 1.9 ± 0.7×105. The

healthy cells were relaxed and looked like smooth muscle cells with a size of approximately

10×50-100 m (Fig. 1), while the damaged cells were hyper-contracted having a rough surface.

At this stage, the cells were generally resistant to the formation of GΩ seals with a patch pipette.

To be used in patch-clamp studies, the final digestion process using papain is essential. Once a

GΩ seal was formed, it was fairly easy to break the patch membrane with suction or an electrical

pulse (1.5 V applied to the patch for various durations) combined with a suction of about 50

mmHg.

Animal preparation: This protocol has been used for isolating atrial cells from rats having a body

weight ranging 150 to 350 g, with the optimal body weight of about 250 g. To obtain the best

yield of cells, minor adjustments should be made with the perfusion volume of the papain

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containing solution based on body weight. Younger rats not only make the cannulation process

difficult but also decrease the yield of cells. On the other hand, older rats will need more time

for perfusion with papain or the digestion of the tissue will be poor. The dose of anesthetic is

important, and what we used was sufficient to put the rat deep enough for the procedure and yet

have the rat still breathing before removing the heart. Heparin injection is also very important

for a successful perfusion in the next step because blood clotting in the coronary blood vessels

will lead to poor digestion.

Cannulation: The heart should not be cannulated immediately after it is removed. More

consistent results were obtained if the heart became quiescent in an ice-chilled solution before

cannulation. Removal of peripheral tissues from the heart should be minimized. Damaging the

tissue between the ventricles and the atria on the back of the heart during dissection is a major

reason for a failed perfusion. After the cannula has been tied to the aorta, only two pieces of fat

tissue and the lungs should be removed. Bubbles in the perfusion tubing should always be

avoided (all tubing should be pre-filled).

To prevent the residual blood inside the left ventricle from interfering with enzymatic

digestion, the end of the cannula was inserted into the left ventricle and the residual blood was

flushed out before the aorta was tied to the cannula. During the perfusion, the increased pressure

in the left ventricle would close the atrioventricular valve and open the aortic valve. The

perfusate in the left ventricle would then enter the coronary arteries through the aorta and perfuse

throughout the heart.

Enzymatic digestion: The most unique aspect of our protocol is perfusing papain in the primary

digestion instead of collagenase. Carrying out the perfusion at room temperature instead of 37C

reduces enzymatic activity and the effect of temperature fluctuation, both are higher at higher

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temperatures. In addition, the milder activity of papain also allows the duration of perfusion to

be lengthened so that slight variation in the concentration or duration would not affect the result

as markedly as in the conventional Langendorff-perfusion process. When papain from different

vendors (Fluka, Sigma or Worthington) was used in our experiments, similar results were

obtained. This suggests that the purity requirement for papain is not as strict as for collagenase.

After papain perfusion, the atria should swell, become pale and flaccid. Also, for a period

of time during the perfusion, the perfusate coming out of the heart will become “sticky”. If the

color of the atria remains pink and there is little change in the size of the atria after perfusion, it

indicates that the perfusion/digestion is poor. This could be due to problems related to any of the

issues discussed above in the animal preparation or cannulation steps. Or, it could be caused by

the perfusate leaking out. If these indications are observed, the atria should be cut into pieces

smaller than what would normally have been done before the second digestion step.

The collagenase digestion is carried out in a nominally Ca2+-free solution (no added Ca2+),

not a solution with 50 M Ca2+ as in other published methods (2). It also appears that the effect

of collagenase digestion is not that crucially dependent on the type or lot of collagenase. Usually,

type II collagenase from Worthington was used in the procedure. When we tried other

collagenases (such as type IV from Worthington, types I and V from Sigma, or types B and D

from Boehringer Mannheim) similar outcomes were achieved. We suggest using 2 mg/ml of

both collagenase and BSA in the digestion solution because it gave us the best result with type II

collagenase from Worthington (478 units/ml). These concentrations were also used to obtain

reasonably good yields with other collagenases tested although it might be necessary to adjust

them for optimization with each collagenase.

Notes on solutions: BDM is used to prevent cardiomyocyte contraction in a reversible way

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because it reduces both Ca2+ currents and the responsiveness of the myofilaments to Ca2+ (3). In

order to prevent the cell contraction due to sudden influx of Ca2+, or “Ca2+ paradox” (4), we used

taurine in the solution (5) and carried out a stepwise restoration of the Ca2+ concentration (6).

The Glutamate-K solution is designed to prevent Ca2+ influx and Na+ loading - reducing

cytosolic Na+ will help prevent Ca2+ influx via the Na+/ Ca2+ exchanger during the Ca2+

restoration process. ATP used in the Glutamate-K solution helped increase the yield of viable

cells (7). We adopted the practice of using low concentrations of divalent cations during the

papain digestion to “loosen” the intercellular connections (8). However, in our protocol, it is

important to raise the Mg2+ concentration after papain digestion to improve the yield of viable

cells. It is possible that Mg2+ can preserve the integrity of the cell membrane by preventing the

separation of the glycocalyx, which is superficial to the plasma membrane. It was suggested that

this separation is associated with a large gain of cellular Ca2+ and contracture upon Ca2+

restoration (9).

Modifications of the protocol for mouse atrial cells and for mouse and rat ventricular cells:

Mouse atrial cells: The same procedure used for isolating rat atrial cells was also used for

isolating mouse atrial cells except that during digestion step-1 the perfusion rate and the amount

of perfusion fluid were half of that used for rat atrial cells. We also used a smaller infusion set

for cannulating mouse heart.

Mouse and rat ventricular cells: The steps for heart isolation and perfusion were the

same as that used in isolating rat atrial cells. The steps after perfusion were modified and they

are discussed below.

Collagenase digestion: The ventricular tissue (rat right ventricular wall or whole mouse

heart) was cut into about 2x2 mm chunks and put into a 25 ml glass flask containing 10 ml of the

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digestion solution. The digestion solution was BDM-HEPES solution containing type II

collagenase (Worthington, 239 units/ml), hyluronidase (908 units/ml) and DNase-I (30 units/ml).

DNase-I was prepared as a stock solution of 900 units/150l of the BDM-HEPES solution that

contained 0.1 mg/ml BSA. Incubation was carried out for 30 minutes at 32C in a shaking water

bath (GCA corporation/Precision Scientific Group, USA) at a speed of around 30 oscillations per

minute.

Trituration: After the incubation, the tissue pieces were transferred into a 15 ml plastic

tube containing 5 ml Glutamate-K solution and triturated 10 – 20 times with a wide-bore plastic

transfer pipette (Cat No 202, SAMCO Scientific Corporation). The cells in the suspension were

transferred into a 25 ml flask. Then, 5 ml of the Glutamate-K solution was added to the tube

containing the tissue and triturated again. The cells in suspension after trituration were, again,

transferred in the flask that contained the cells from the first trituration. Such trituration

procedure was repeated for another two times and cells from all 4 batches were pooled together

to make roughly 20 ml of cells. The pooled cells were filtered through a plastic mesh (openings

300~500 microns, Small Parts Inc.) and collected in a 25ml flask. After resting for 10 minutes,

the upper layer was removed, leaving viable cells at the bottom to use in the experiment.

Depleting the Ca stores: This step was designed for isolating ventricular cells to reduce the

number of contracted cells. The upper layer of the mixture was removed at the end of the 20

minutes and 5 ml of the “depleting solution” was added to the flask. The depleting solution was

the BDM-HEPES solution with the addition of 1 mM Na2ATP, 1 mM MgSO4 and 1 M

Thapsigargin. This mixture was allowed to rest at room temperature for 30 min. Next, 1 ml of a

50 mM caffeine stock solution (prepared in depleting solution) was added to the mixture and the

mixture was left at room temperature for an additional 10 minutes. The cells were then washed

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twice at 10 minute intervals with the depleting solution in a 15 ml plastic tube (by removing the

upper layer and adding 5 ml of the depleting solution and mixing by gently inverting the tube).

Restoring Ca: The same procedure was used as that for rat atrial cells. By adding 0.1 M

CaCl2 every 7 minutes, the Ca2+ concentration was adjusted, stepwise, to 0.05, 0.1, 0.2, 0.3, 0.5,

0.7 and 1.0 mM. Cells were stored at 4C and used within 5 hours.

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Circulation Research 69: 1280-1292, 1991.

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