engleski

A method for isolation of functional human ventricular myocytes from fresh epicardial biopsies

Most of basic research findings in the field of cardiac cell physiology rely on data gathered in cells obtained from various animal models or tissues other than human ventricle (atrial myocardium or derived from iPS cells). Although these approaches offer possibilities of mechanistic investigations, they still have many limitations leading to difficult “translatability” to real-life human and clinical situations. We developed a protocol for isolation of functional cardiac myocytes from small epicardial biopsy samples of human left ventricle obtained during open-chest coronary artery-bypass grafting surgery. Biopsy was performed in patients undergoing such surgery who gave their informed consent. Biopsy samples weighing 5-10 mg were immersed in ice-cold BDM-supplemented cardioplegia solution, cut to 350 m pieces and incubated in Joklik medium containing trypsin and Liberase TM enzymes, and gently shaken for approximately 60 min at 37 C. Viable rod-shaped cardiomyocytes appear after 45 min of digestion. Afterwards, 1 ml of cell suspension is transferred to enzyme-blocking solution and settled for 10 min. This step is repeated every 5 min until tissue pieces become completely dissolved. Joklik medium is then replaced with Tyrode solution and calcium is gradually added to 1.2 mM. In the end, calcium-tolerant cardiomyocytes were loaded with Fura-2AM and were able to contract upon electrostimulation and exhibit stable calcium transients. Cardiomyocytes obtained by the described procedure could be used for other purposes, such as immunocytochemistry, patch clamping, contractility assessment, etc. The procedure of sample collection is straightforward, safe for patients and is not limited to specific type of cardiac surgery.

human cardiomyocytes; epicardial biopsies; isolation

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