Abstract
Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4–8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.
Highlights
Single cells, isolated from various tissues, have been a foundation for the development of mechanistic insight into many organ systems, promoting the understanding of physiology, pathology, and therapeutic interventions
We show that live tissue storage [12] can be combined with slice-based CM isolation [13] to yield CM from the same donor heart over multiple days, circumventing the adverse remodeling in transcript levels, microscopic structure, and contraction and relaxation kinetics that are otherwise seen in isolated CM over the same period of time
We present a method of extending the use of cardiac tissue for the isolation of primary ventricular and atrial CM past the day of organ excision
Summary
Single cells, isolated from various tissues, have been a foundation for the development of mechanistic insight into many organ systems, promoting the understanding of physiology, pathology, and therapeutic interventions. In experimental cardiac research, CM isolated from human and animal hearts have become indispensable over the 50 years since the first description of Langendorff perfusion-based enzymatic CM isolation [1]. Primary adult CM isolation is performed after sacrificing the experimental animal. While every successful isolation yields millions of CM, most of these cells can be used for functional research only over a relatively short time period. For each set of single CM experiments, an animal is sacrificed, each heart contains enough CM for tens, if not hundreds, of functional experiments
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