A novel method to extend viability and functionality of living heart slices

Figures

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  Figure 1. Overview of the novel method for heart slice preparation and storage. (1) After excision, the porcine heart is retrogradely perfused via the aorta with 100 ml of ice-cold cardioplegia solution. (2) Transmural tissue blocks (1.3 cm × 1.3 cm) are dissected from the left ventricle using a metal razor blade. (3) Tissue blocks are mounted on the tissue holder of a high precision vibrating microtome with epicardium down, using surgical glue Histoacryl. (4) 300 µm thick slices are prepared using a ceramic blade at a cutting speed of 0.04 mm/s in ice-cold oxygenated Tyrode's solution with 10 mM BDM. (5) Slices are incubated for at least 20 min at RT in 100 μm nylon mesh cell strainers to recover. (6) Sterile transparent plastic triangles are bond perpendicularly to muscle fibre direction on opposite side of each slice. (7) After plastic triangle attachment, the resting length of each slice is measured using a caliper. (8) For storage experiments, slices bond to plastic triangles are pinned in PDMS-coated dishes at resting length and are subsequently stored in hypothermic conditions. (9) Assays used to validate preservation of slices include contractility, LDH activity, electrical…

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  Figure 2. Contractility of porcine heart slices stored at 4°C in Tyrode's solution, Tyrode's solution supplemented with BTS and Tyrode's solution supplemented with BDM. (A) Representative contraction traces from freshly prepared porcine slices (day 0) and porcine slices stored for 2 days at 4°C in the 3 different solutions. (B) Contractility of porcine slices over the 2-days storage period in the 3 solutions. Slices were field stimulated at 1 Hz pacing frequency. Bars represent means ± SEM. Black dots represent individual data points (n = 5–9 slices/3–4 hearts). $ p < 0.05 compared to day 0; *p < 0.05, **p < 0.01 between two groups (one-way ANOVA with Tukey's multiple comparisons test).

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  Figure 3. Contractile function of porcine heart slices after storage at 4°C in Tyrode's solution supplemented with BDM over a 6-day period. (A) Representative contraction traces from freshly prepared porcine slices (day 0) and porcine slices after storage at 4°C in BDM-supplemented Tyrode's solution for 1, 2, 3 and 6 days. Right: contraction trace showing parameters analyzed. (B) Contractility of porcine slices over storage time. (C,D) Time to contraction peak and time to 100% relaxation. Bars represent means ± SEM. Black dots represent individual data points (n = 12–16 slices/4 hearts). (E) Contractility produced by porcine slices under stepwise stretch and 1 Hz stimulation (n = 16 slices/4 hearts at day 0; n = 12 slices/4 hearts at day 1, 2, 3 and 6; means ± SEM).

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  Figure 4. Viability of porcine heart slices stored at 4°C in BDM-supplemented Tyrode's solution over 6 days. LDH release in the media is expressed as percentage (%) of positive control (LDH levels in media from slices lysed with the lysis solution) normalized to slice weight (in mg). Bars represent means ± SEM. Black dots represent individual data points (n = 6–10 slices/3–4 hearts). $ p < 0.05 compared to day 0 (one-way ANOVA with Tukey's multiple comparisons test).

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  Figure 5. Field potential duration in porcine heart slices stored at 4°C in BDM-supplemented Tyrode's solution over 6 days. (A) Representative field potential traces recorded during point stimulation at 1 Hz frequency in freshly prepared porcine slices (day 0) and porcine slices stored at 4°C in BDM-supplemented Tyrode's solution for 1, 3 and 6 days. FPD: field potential duration. (B,C) Field potential duration at 1 Hz and 2 Hz pacing frequency. Bars represent means ± SEM. Black dots represent individual data points (n = 14–24 slices/4 hearts). $$ p < 0.01, $$$ p < 0.001, $$$$ p < 0.0001 compared to day 0; *p < 0.05, **p < 0.01 between two groups (one-way ANOVA with Tukey's multiple comparisons test).

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  Figure 6. Sarcomere length in porcine heart slices is preserved over 6 days during storage at 4°C in BDM-supplemented Tyrode's solution. (A) Confocal microscopy images of slices stained for α-actinin briefly after preparation (day 0) or after storage at 4°C in BDM-supplemented Tyrode's solution for 1, 2, 3 and 6 days. Scale bar: 10 µm. (B) Intensity profile of the user-drawn path (red line in day 1 image) used to quantify sarcomere length. (C) Mean sarcomere length of fresh slices (day 0) and slices stored at 4°C in BDM-supplemented Tyrode's solution for different duration. Bars represent means ± SEM. Black dots represent individual data points (n = 6 slices/3 hearts; p > 0.05; one-way ANOVA with Tukey's multiple comparisons test).

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  Figure 7. Quantification of stromal cells in porcine heart slices stored at 4°C in BDM-supplemented Tyrode's solution over a 6-day period. Representative confocal images of slices stained for vimentin (green) and nuclei (red). Scale bars: 100 µm. Bar graph summarizing quantification of vimentin in slices stored for different duration. Bars represent means ± SEM. Black dots represent individual data points (n = 6 slices/3 hearts; p > 0.05; one-way ANOVA with Tukey's multiple comparisons test).