A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice.

Abstract

The aim of this study was to establish a simple and reproducible antegrade perfusion method for isolating single viable mouse heart cells and to determine the standard practical protocols that are appropriate for mice of various ages. Antegrade perfusion was performed by injecting perfusate from near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. This could thoroughly perfuse the myocardium through the coronary circulation. All procedures were carried out on a prewarmed heater mat under a microscope, which allows for the processes of injection and perfusion to be monitored. With appropriate adjustment of the size of the injection needle, the composition and amount of enzyme solution and the perfusion flow rate, this antegrade perfusion method could be applied to the hearts of neonatal to aged mice. We examined the morphological characteristics and electrophysiological properties of the isolated ventricular and atrial myocytes and found that these cells were mostly identical to those obtained with the traditional Langendorff‐based retrograde perfusion method. Interstitial nonmyocytes, such as cardiac progenitor cells, were also isolated simultaneously from the supernatant fraction of the centrifugation, similar to the retrograde perfusion method. The results suggest that single heart cells can be well isolated with high degree of quality by the present antegrade perfusion method, regardless of the age of the mouse.