Cardiomyocyte Isolation from 300µm rabbit left atrial & left ventricular slices

Abstract

(from Consecutive-Day Ventricular and Atrial Cardiomyocyte Isolations from the Same Heart: Shifting the Cost–Benefit Balance of Cardiac Primary Cell Research, Greiner et al, 2022)

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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

Method

After excision, the heart was Langendorff perfused for ~1 min (until cessation of contractions) with warm (37 °C) modified Tyrode solution (flow rate 20 mL/min) containing (in mM): 138 NaCl, 0.33 NaH2PO4, 5.4 KCl, 2 MgCl2, 10 HEPES, 10 glucose, 0.5 CaCl2, and 30 2,3-butanedione monoxide; pH 7.3, 300 mOsm. This step can be omitted when processing tissue that cannot be perfused.

The heart was then dissected in cold (4 °C) modified Tyrode solution; the left ventricular free wall, left atrial free wall, and left atrial appendage were used for further processing. The tissue was cut into blocks with an X/Y size of up to 1 × 1 cm2 (ventricle)/0.5 × 0.5 cm2 (atria). These were embedded in 4% low melting point agarose (Carl Roth, Karlsruhe, Germany) prepared with modified Tyrode solution, cooled to 4 °C, and mounted on the stage of a vibratome (7000-smz-2 with 7550-1-SS blade, both Campden Instruments Ltd., Loughborough, UK) using cyanoacrylate-based low-viscosity glue. During mounting, care was taken to visually estimate the prevailing orientation of CM within the slice, and to mount the tissue so that the cutting plane would be parallel to the epicardial surface of the tissue block. The tissue was submerged in ice-cold modified Tyrode solution bubbled with O2, and sliced into 300 µm thick sections at a vertical blade vibration frequency of 60 Hz (ventricle) and 80 Hz (atria), an amplitude of 1.5 mm, and an advancement velocity of 40 µm/s (ventricle) and 10 µm/s (atria). Ventricular tissue was freshly sliced from tissue chunks on each day before CM isolation, whereas atrial tissue was sliced on the day of heart excision and the slices were stored until CM isolation. All tissue was stored in modified Tyrode solution at 4 °C and under room atmosphere, for up to three days.