Direct isolation of primary cells from tissues and organs allows for the maintenance of important cell characteristics and properties for in vitro studies and a plethora of biomedical applications. Dissociation of cells from the organ of interest is possible due to the enzymatic activity of collagenases. The choice and the dose of these enzymes is the critical step to obtain the maximum number of cells with intact structure and function. In this contest, Abiel collagenases class I (Col G) and class II (Col H) were synthesised using recombinant DNA technologies and their ability to degrade collagen in cell isolation from different tissues was tested. Examples of cells isolated with these enzymes include Langerhans islets, hepatocytes, chondrocytes, osteoblasts, fibroblasts, and stem and retinal cells that be used for in vitro studies. In this contest, primary cardiomyocytes represent a perfect murine model to investigate numerous heart diseases. Herein, a protocol of cardiomyocyte extraction from rat heart, using a combination of Abiel collagenases supplemented with thermolysin protease, is proposed. The structure and the viability of isolated cells were tested over time by optical and fluorescence microscopy and viability assays. Further cellular structure characterisation was performed by western blot analysis, using specific cardiomyocytes markers. Isolation of viable primary heart cells with unaltered properties and functionality can potentially provide in vitro models to study heart function, arrhythmias, long Q-T syndrome and cardiotoxicity.