Download Isolation of Mouse Cardiac Myocytes with Blendzyme 4

Isolation of Mouse Cardiac Myocytes with
Blendzyme 4
Prof. K. Sipido, Laboratory of Experimental Cardiology
Prof. K. Mubagwa, Center for Experimental Surgery and Anesthesiology
University of Leuven, Leuven, Belgium.
From:
Sipido KR, Callewaert G, Carmeliet E. Inhibition and rapid recovery of Ca2+ current during Ca2+
release from sarcoplasmic reticulum in guinea pig ventricular myocytes. Circ Res 1995;76(1):1029.
Online reference
Mubagwa K, Stengl M, Flameng W. Extracellular divalent cations block a cation non-selective
conductance unrelated to calcium channels in rat cardiac muscle. J Physiol 1997;502 (Pt 2):235247.
Online reference
Method
The technique is based on perfusion of the heart through the coronary system, by cannulating the
aorta. The method consists of five basic steps:
1. The heart is washed with a normal Tyrode's solution during which it is beating spontaneously
([Ca2+] = 1.8 mM).
2. The perfusion is switched to a nominally Ca-free Tyrode's solution, and the heart becomes
quiescent.
3. The heart is perfused with the enzyme solution.
4. The enzyme solution is washed with a low Ca2+ Tyrode's solution ([Ca2+ = 0.18 mM).
5. The tissue is dissociated by mincing and shaking in this same low Ca2+ solution. Cells are
washed and resuspended in Tyrode's solution.
Methods Detail
Liberase Blendzyme 4, concentration: 0.1 mg/ml
After cannulation of the aorta, the heart is mounted on a Langendorf setup, with either constant
pressure perfusion (80 mm Hg, flow rate is variable, but at least 5 ml/min), or with constant flow
rate (5 ml/min, pressure is variable).
For washing blood we use a Tyrode's solution, in mM: NaCl 137, KCl 5.4, MgCl2 0.5, CaCl2 1.8,
Na-HEPES 11.8, glucose 10; pH 7.40. This step takes less than 5 minutes.
Next, the heart is perfused with Ca-free Tyrode, prepared in ion-free and sterile water purchased
from the hospital pharmacy, in mM: NaCl2 130, KCl 5.4, KH2PO4 1.2, MgSO4 1.2, Na-HEPES 6,
pH 7.20. This steps lasts approximately 7 minutes in small mammals.
The next step is perfusing with enzyme, dissolved in the Ca-free Tyrode, and this solution is
usually recirculated. For small mammals the duration is from 7 to 10 minutes. During this time the
tissue becomes soft and friable. In the constant pressure system, the flow rate can sometimes
double due to opening of capillary shunt flow.
On the last step, the enzyme is washed out with low Ca solution, i.e., the Ca-free Tyrode to which
0.18 mM CaCl2 was added. This step lasts from 10 to 15 minutes. The ventricles or atria are
removed, and gently minced and agitated in a beaker until a cloudy cell suspension is obtained.
Cells are allowed to settle and are washed two times. Next the solution is gradually replaced with
the normal Tyrode's solution. Cell yield and viability are judged in this Ca-containing solution.