Isolation of adult mouse and rat cardiomyocytes

The mouse and rat cardiomyocyte isolation protocols share the same experiment device settings:

Langendorff perfusion system with ~ 90 cm water pressure. Using the Double Baker Warming Coil (Harvard Apparatus, Cat No. PY8 50-8382) to switch between the perfusion buffer and enzyme digestion buffer.
Clean the isolation rig (including recirculation tubes) with 70% ethanol 1 time and 2 times with autoclaved ddH₂O. Totally empty the system after each wash.
Turn on 37 ̊C water bathes and open the oxygen tank supply to bubble the perfusion solutions.

Part I: Adult mouse cardiomyocyte isolation.

Buffers:

Mouse cardiomyocyte isolation buffer (pH 7.4):

Chemical	Final Con. (mM)
NaCl	113
KCl	4.7
MgSO₄ x 7H₂O	1.2
NaH₂PO₄	0.6
KH₂PO₄	0.6
HEPES	10
Glucose	10
Taurine	5

Digestion buffer (50 ml):

Myocyte Isolation Buffer + Collagenase (300 U/ml) + Hyaluronidase (0.5 mg/ml) + Blebbistatin (10 µM)

Stop digestion buffer (50 ml):

Cardiomyocyte Isolation Buffer + BSA (2.5%) + CaCl₂ (0.1 mM) + Fetal Bovine Serum (5%) + Blebbistatin (10 µM)

Procedures:

Fill the system with mouse cardiomyocyte isolation buffer (left tube) or enzyme digestion buffer (right tube).
Euthanize the mouse by cervical dislocation. Spay the mouse chest with 70% ethanol.
Open the chest cavity and take out the heart with about 2 mm of aorta quickly. Put the heart in the mouse cardiomyocyte isolation buffer on the ice.
Cannulate the heart with a 23 g needle covered by a thin plastic tube (to prevent a loose tie) and tie the aorta to the cannula with 6-0 silk thread. The tip of the cannula should be just above the aortic valve. The total time to cannulate the heart should be less than 1 min.
Perfuse the heart with mouse cardiomyocyte isolation buffer (0 Ca²⁺, 2-3 mL/min) for 5 min.
Switch to digestion buffer (300 U/mL collagenase II + 0.5 mg/mL hyaluronidase) for 5 min. Add 40 µL CaCl₂ (100mM stock) to the digestion buffer, circulate the digestion buffer.
Perfuse digestion buffer for 20-30 min.
Take down the heart, remove atria, tear apart the heart tissue in digestion buffer using forceps.
Leave the tissue in 5 mL digestion buffer for 5 min. Pipette up and down 5-6 times in a 1 mL pipette tip. Note: cut the end of the 1 mL tip to approximately 2 mm diameter to make sure that the tissue easily goes into the tip.
Collect the isolated cells, add 5 mL stopping buffer in a 15 mL tube. And repeat step 9.
Combined all the isolated cells. Centrifuge at 300 rpm for 20 s or let the cells settle down for 5 min.
Remove supernatant, add 10 mL new stop buffer to suspend the cells.
Add 10 µL CaCl₂ (100 mM) for 4 times with 5 min interval between each addition for gradual Ca²⁺ restoration to avoid the high Ca²⁺ concentration stress. Each time, mix the solution by gently pipetting up and down 3 times.
Centrifuge down cells at 150 g for 20 s or let the cells settle down for 5-10 min.
Resuspend cells in the appropriate buffer or medium for experimental use.

Part II: Adult rat cardiomyocyte isolation.

Buffers:

Rat cardiomyocyte isolation buffer (pH 7.4, adjusted with NaOH) with or without 1 mM Ca²⁺, filtered through 0.2 μm filter:

Chemical	Final Con. (mM)
NaCl	118
KCl	4.8
HEPES	25
KH₂PO₄	1.2
MgSO₄ x7H₂O	1.2
CaCl₂ x 2H₂O*	1.0
Glucose	11

*for the enzyme digestion buffer, no Ca²⁺ added.

Digestion buffer:

In 20 mL Ca²⁺ free isolation buffer adding 6, 500 U collagenase II and 12 mg hyaluronidase.

Stop digestion buffer:

2% BSA in 0.1 mM Ca²⁺ isolation buffer filtered through 0.2 μm filter.

Procedures:

1. Fill the system with 1 mM Ca²⁺ perfusion buffer (left) or Ca²⁺ free buffer (right).

2. i.p. inject with pentobarbital sodium 1mL (50mg/mL) to anesthetize the rat (~ 300 g) and heparin 0.5 mL (10,000 U) to prevent blood clotting.

3. Spay the rat chest with 70% ethanol. Open the chest wall, cut the ribs and quickly remove the heart and 3-5mm of attached aorta. Put the heart in 1 mM Ca²⁺ isolation buffer on ice.

4. Cannulate the heart with an 18 G needle (covered with a plastic tube) connected to a 5 mL syringe filled with 1mM Ca²⁺ perfusion buffer and tie the aorta to the cannula with 4-0 silk thread. The tip of the cannula should be just above the aortic valve. Press buffer into aorta to remove blood, hang the heart on the isolation rig.

5. Perfuse with 1 mM Ca²⁺ perfusion buffer for 5 min (1-2 drops/sec).

6. Switch to Ca²⁺-free perfusion buffer for 5 min.

7. Add 20 mL concentrated digestion buffer (~6,500 U collagenase II and 12 mg hyaluronidase) to the Ca²⁺-free perfusion buffer remains in the tube (~ 60 mL).

8. Put the heart in a 50 mL beaker and set up recirculation of the digestion buffer. Submerge the heart in the digestion buffer.

9. After 5 min, add 150 uL 100 mM CaCl₂ 3 times at 1 min intervals into the Ca²⁺-free buffer.

10. Monitor heart digestion: it should swell and look soft, pinkish, but not pale white; the flow rate will increase during perfusion as the heart swells. Total digestion time is about 40-60 min.

11. Take down the heart, remove the atria and aorta, cut the ventricles into small chunks (~2 mm square)

12. Add 15 mL enzyme digestion buffer (from the 80 mL digestion buffer) and gently swirl in a 37 °C water bath for 5 min.

13. After first swirl, do not pipette the chunks, gently pipette the first swirl solution which contains mostly dead cells and discard it. Add 15 mL enzyme buffer, swirl the 2nd time for 5 min and pipette the chunks up and down to disperse the cardiomyocytes. Collect the dispensed cells in a 15 mL tube. Add 15 mL enzyme buffer to rest of the chunks for the 3rd digestion. Repeat for about 3-4 times till the chunks become white and only connective tissue is left.

14. Combined all the isolated cells. Centrifuge 150 g for 20 s. Resuspend cells in 20 mL stop digestion buffer placing 10 mL in each of two 15 mL tubes. Add 25 uL 100 mM CaCl₂ in each tube 3 times at 5 min intervals, to gradually restore the Ca²⁺.

15. Centrifuge 150 g, 20 s. Resuspend cells in in the appropriate buffer or medium for experimental use.