Biomimetic cardiac tissue culture model (CTCM) to emulate cardiac physiology and pathophysiology ex vivo

Figures

• 

  Fig. 1. Illustration of the cardiac tissue culture model (CTCM). a The tissue slices are attached to 7-mm diameter support rings shown in blue within the culture chamber of the device. The culture chamber was separated from the air chamber by a thin, flexible silicone membrane. Between each chamber, a sealing gasket was placed to prevent leaks. The cover for the device contains graphite electrodes that provide electrical stimulation. b Schematic illustration of tissue oversizing apparatus, ring guide, and support ring. Tissue slices (brown) are positioned on the oversizing device, and the ring guide is placed in the grove on the outer edge of the apparatus. Using the guide, the support ring coated with histoacryl glue is carefully placed on the heart tissue slice. c A diagram depicting the timing of the electrical stimulation in relation to the pressure within the air chamber controlled by the programmable pneumatic driver (PPD). A data acquisition device was used to synchronize electrical stimulation using a pressure probe sensor. When the pressure within the culture chamber reaches a specified threshold, an impulse signal is sent to the C-PACE-EM device to induce electrical…

• 

  Fig. 2. Characterization of the CTCM and evaluation of the heart slice stretches over time in culture. a Representative traces of the air chamber pressure, fluid chamber pressure, and tissue movement measurements verified that the air chamber pressure changes the fluid chamber pressure, which induces a corresponding tissue slice movement. b Representative traces of the percent stretch (blue) of the tissue slices correspond with the percent stretch rate (orange). c The heart slice movement measured was consistent with the measured movement speed. (d) Representative trace of the heart slice cycle movements (blue line) and speed (dotted orange line). e Quantification of the cycle time (n = 19 slices/group from different pigs), contraction time (n = 19 slices/group), relaxation time (n = 19 slices/group from different pigs), tissue movement (n = 25 slices/group from different pigs), peak contraction speed (n = 24 (D0), 25 (D12) slices/group from different pigs); and peak relaxation speeds (n = 24(D0), 25 (D12) slices/group from different pigs). Two-tailed Student t-tests revealed no significant difference in any of the parameters. f Representative trace for strain analysis of a tissue slice…

• 

  Fig. 3. Mechanical stimulation partially improves tissue slice viability and structural integrity after 12 days in culture. a Bar graph shows quantification of the MTT viability of fresh heart slices (D0) or heart slices culture for 12 days either in static culture (D12 Ctrl) or in CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) slices/group from different pigs, one way ANOVA test is performed; ####p < 0.0001 compared to D0 and **p < 0.01 compared to D12 Ctrl). b Representative immunofluorescence images for troponin-T (green), connexin 43 (red), and DAPI (blue) for freshly isolated heart slices (D0) or heart slices cultured for 12 days under static conditions (Ctrl) or CTCM conditions (MC) (Scale bare = 100 µm). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) slices/group each from different pig, one-way ANOVA test is performed; ####p < 0.0001 compared to D0 and ****p < 0.0001 compared to D12 Ctrl). c Representative images (left) and quantification (right) for heart slices stained with Masson’s trichrome stain (Scale bare = 500 µm) (n = 10 slices/group each from different pig, one-way ANOVA test is performed;…

• 

  Fig. 4. Combining mechanical and humoral stimulation (MT condition) preserve the heart slices for 12 days. a Schematic representation of the experimental design depicting the four culture conditions that were used to assess the effect of combining mechanical stimulation and T3/Dex addition to the culture media over the course of 12 days. b Bar graph shows quantification of viability 12 days post culture in all 4 culture conditions (Ctrl, TD, MC, and MT) compared to fresh heart slices (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD and D12 MT), 12 (D12 MC) slices/group from different pigs, one-way ANOVA test is performed; ####p < 0.0001, ###p < 0.001 compared to D0 and **p < 0.01 compared to D12 Ctrl). c Bar graph shows the quantification of glucose flux 12 days post culture in all 4 culture conditions (Ctrl, TD, MC, and MT) compared to fresh heart slices (D0) (n = 6 slices/group from different pigs, one-way ANOVA test is performed; ###p < 0.001, compared to D0 and ***p < 0.001 compared to D12 Ctrl). d Strain analysis plot for fresh (blue), day 12 MC (green), and day 12 MT (red) tissue across the ten regional points of the tissue slice (n = 4 slices/group, one-way ANOVA test is performed; no…

• 

  Fig. 5. Cardiac tissue structure in slices cultured for 12 days under MT conditions is similar to fresh heart slices. a Representative immunofluorescence images for troponin-T (green), connexin 43 (red), and DAPI (blue) for freshly isolated heart slices (D0) or heart slices cultured for 12 days under all four culture conditions (Scale bar = 100 µm). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0 and D12 Ctrl), 5 (D12 TD, D12 MC and D12 MT) slices/group from different pigs, one-way ANOVA test is performed; ####p < 0.0001 compared to D0 and *p < 0.05, or ****p < 0.0001 compared to D12 Ctrl). b Representative images and quantification for heart slices stained with Masson’s trichrome stain (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, and D12 MC), 9 (D12 MT) slices/group from different pigs, one-way ANOVA test is performed; ####p < 0.0001 compared to D0 and ***p < 0.001, or ****p < 0.0001 compared to D12 Ctrl). Error bars are representative of the Mean ± SD.

• 

  Fig. 6. Induction of pathological overstretching in heart slices cultured in CTCM device. a Representative traces of the air chamber pressure, fluid chamber pressure, and tissue movement measurements verified that the air chamber pressure changes the fluid chamber pressure, which induces a corresponding tissue slice movement. b Representative traces of the percent stretch and stretch rate of normal stretch (orange) and overstretched (blue) tissue slices. c Bar graphs showing quantification of the cycle time (n = 19 slices/group from different pigs), contraction time (n = 18–19 slices/group from different pigs), relaxation time (n = 19 slices/group from different pigs), tissue movement amplitude (n = 14 slices/group from different pigs), peak contraction speed (n = 14 slices/group from different pigs), and peak relaxation speeds (n = 14 (D0), 15 (D6) slices/group from different pigs), Two-tailed Student t-tests revealed no significant difference in any of the parameters, demonstrating that these parameters remained consistent over 6 days of overstretching culture. Error bars are representative of the Mean ± SD.

• 

  Fig. 7. Overstretching in CTCM induces pathological hypertrophy. a Bar graph quantification of NT-ProBNP concentration in culture media from heart slices cultured under MT normal stretch (Norm) or overstretching (OS) conditions (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, and D4 MTOS) slices/group from different pigs, Two-way ANOVA is performed; **p < 0.01 compared to normal stretch). b Representative images for heart slices stained with troponin-T and WGA (left) and cell size quantification (right) (n = 330 (D6 MTOS), 369 (D6 MTNorm) cells/group from 10 different slices from different pigs, Two-tailed Student t-test is performed; ****p < 0.0001 compared to normal stretch). c Representative images for day 0 and day 6 MTOS heart slices immunolabeled for troponin-T and NFATC4 and quantification of the translocation of NFATC4 to the nuclei of CMs (n = 4 (D0), 3 (D6 MTOS) slices/group from different pigs, Two-tailed Student t-test is performed; *p < 0.05). Error bars are representative of the Mean ± SD.