Dear all,

I'd like to know your opinion about an issue I'm facing during the isolation of adult rat cardiomyocytes (CMs).

The protocol I've been following is as follows:

- Rats are anhestetized and heparinized before heart extraction

- Langendorff perfusion of isolated heart with Krebs solution (119 mM NaCl, 4.7 mM KCl, 0.94 mM MgS04, 1.2 mM KH2PO4, 25 mM NaHCO3, 11.5 mM glucose, 1 mM CaCl2 and equilibrated to ph 7.4) until the flow is clear from blood

- Low calcium buffer perfusion of 5 minutes step (120 mM NaCl, 5.4 mM KCl, 5 mM MgSO4, 5 mM pyruvate, 20 mM glucose, 20 mM taurine, 10 mM HEPES, 12-14 mM of CaCl2, 5 mM NTA pH-balanced to 7.4)

- A solution similar to the previous one but without NTA and 200 uM CaCl2 addition and with 1 mg/mL of collagenase type II (210 units/mg) and 0.6 mg/mL hyaluronidase, perfused for about 10 minutes

The flow rate is set to 1 drop/second.

- The heart is then removed from the Langendorff system, the ventricles are separated from the atria and minced to get single cells suspension.

- The digestion is completed under the fume hood and after getting the cell pellet, the CMs are resuspended in RPMI, complemented with additional Ca2+ to reach 1.2 mM concentration, BSA (0.2 %), ascorbate (100 mM), creatine (5 mM), carnitine (2 mM), taurine (5 mM), insulin (0.1 uM) and antibiotics.

The issue I've been experiencing is that my cells look pretty good and viable but I can't detect spontaneous contraction or even when paced via field stimulation (range of 8 V).

Could someone help me or suggest some improvements in the protocol?

I'm searching for a more gentle digestion that could improve my cells contraction and I'm doubfull about the Ca2+ gradient throughtout the protocol.

Thank you in advance for the help!