Simplified Method for the simultaneous isolation of viable cardiomyocytes and nonmyocytesfrom the adult mouse heart

Cardiovascular diseases (CVDs) have remained the leading cause of global mortality. Of these, one of the main events leading to death is myocardial infarction (MI), followed by cardiac fibrosis. To better understand the disease and its development, various techniques have been developed to create models for studying cardiovascular conditions. Most of the models developed focus on the role of recruited immune cells, with less models available to study the role of cardiomyocytes. One such approach for the isolation of cardiomyocytes and nonmyocytes from the heart of adult mice for in vitro studies is the Langendorff isolation. This current technique relies on retrograde aortic intubation of the heart using a specialized Langendorff apparatus. The high cost and extensive training requirement associated with this device poses an obstacle to researchers, particularly in newly established laboratories. Hence, this study aims to provide an alternative Langendorff-free method for cardiomyocyte and nonmyocyte cardiac cell
isolation through enzymatic digestion and manual operation. Briefly; immediately after sacrifice, the inferior vena cava and descending aorta was cut off and the mouse heart is perfused by EDTA buffer injected into the right ventricle. The aorta was then clamped using a hemostatic clamp followed by the extraction of the heart and placed while still clamped on cold PBS. Afterwards, the heart is further perfused with EDTA buffer slowly injected directly into the left ventricle. The approach is repeated with PBS and then with a collagenase IV/dispase solution to isolate the cells followed by a stop buffer, an FBS solution. The heart is then gently teased apart into smaller pieces followed by filtration through a cell strainer. Cardiomyocytes and nonmyocyte from the cell suspension Abstract for 4th Edition of Cardiology World Conference 2023 Valencia, Spain were separated and collected through gravity settling. Isolated cells can then be cultured in their respective media and calcium reintroduction steps can take place with the cardiomyocytes. The isolated cardiomyocytes were observed to have retained their rod-shaped morphology and contractile function. On the other hand, the fibroblasts isolated were able to proliferate up to four passages. The presented findings indicate that the simplified method proposed in this study is effective in isolating viable cardiomyocytes and nonmyocytes from the adult mouse heart but further analysis is necessary to characterize the isolated cells.

Audience Take Away Notes

• Grasp the difficulties associated in cardiac cell isolation
• Learn a new simplified method for cardiac cell isolation
• Identify potential applications of cardiac cell isolation