This protocol has been optimized for the isolation of cardiomyocytes from neonatal Spraue-Dawley rats (1-2 days)

Before starting any procedure, prepare the following reagents and materials:

A) Dissociation buffer

116 mM NaCl, 20 mM Hepes, 0.8 mM Na2HPO4, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4,

adjust pH to 7.35.

B) Enzyme buffer

Dissociation buffer plus 0.6 mg/ml of pancreatin and 0.4 mg/ml Collagenase Type II.

C) Plating media

Ham’s F10 with 10% horse serum and 5% charcoal- stripped fetal bovine serum (FBS) containing 100 μM bromo-deoxyuridine, 100 μg/L penicillin and streptomycin. Warm at 37ºC.

D) Pre-coated plates

Plates pre-coated with 1% gelatin in PBS place on the plate for about 1-2 minutes. Aspirate off gelatin, and let the plate drie for 60 minutes. 

Procedure:

1. Pre-refrigerate ut to 3 pups at 4ºC for 20 min.

2. Place inside the hood: 

4-10 cm² tissue culture plates. 

One 100 ml beaker with 70% ethanol. 

One 500 ml beaker with glove lining for carcasses.

One rectangular dish full of ice. 

15 ml of dissociation buffer into a 10cm² plate and keep solution chilled on the ice. 

10 ml of enzyme solution into another 10 cm² plate and keep cool on ice.

Into a 10cm² plate: scissors and small forceps. 

Into a second plate: micro-scissors, scalpel with size number 15 blade, and long forceps.

3. Decapitate first pup using large scissors. 

4. Dip the body in 70% ethanol nearly completely (at least to the lower abdomen) 

5. Make one cut with scissors to the left side of the sternum down to the diaphragm and remove the heart with forceps.

6. Place the heart in dissociation buffer on ice while you proceed with the remaining pups.

7. After finish this step, remove instruments and the beakers used from the hood and clean the inside of the hood with 70% EtOH.

8. Place the 10 cm dish containing the hearts onto a 2 cm² gauze piece, and using the long forceps and scalpel, cut the great vessels and atria away from the ventricles of each heart and place the ventricles into the enzyme solution on ice.

9. After all hearts have been trimmed, take the 10 cm² plate containing the hearts and enzyme solution, remove from ice and place onto a gauze. Mince hearts holding the apex or base with the forceps and using the micro-scissors, cut the ventricles transversely and sagittally approximately 7-12 times per heart.

10. After all hearts have been minced use a pipette to deposit the suspension into one of the sterilized tissue dissociation bottles. Close the bottle and place it in the 37ºC water bath, shaking at 80 rpms.

11. Perform serial digestions for the following time periods:

1st - 5 minutes, discard as most cells in the suspension are erythrocytes. 

2nd - 20 minutes 

3rd, 4th - 25 minutes each.

5th - 15 minutes

6th - 10 minutes.

After each digestion, the cell suspension is placed immediately in 2 ml of neonatal calf serum (NCS) that is then centrifuged at 100xg for 5 minutes. Resuspended the pellets in 4 ml of NCS and kept at 37ºC in the incubator.

12. Pool all cell suspensions and centrifuged again for 5 minutes at 100xg. Resuspended in 2ml of Plating media.

13. Passed the suspension through a 70 μm nylon filter and pre-plate for 75 minutes on 3-4 60 mm² plates.

14. After pre-plating, remove the cardiomyocyte-enriched fraction, from each and place fresh media on each and label as non-myocytes. Bring the total volume of the cell suspension to 10 ml/heart. Gently mix the cell suspension and remove an aliquot of cells and place in trypan blue solution (1/4 dilution). Calculate the total yield and plate the cells at the following proportions

For 6 well plates: 750K per well.

For 12 well plates: 200K per well.

For 24 well plates: 100K per well.

For 8 well chamber slides: 30-40K per well.