Viability and Longevity of Human Miniaturized Living Myocardial Slices

Abstract

Living myocardial slices (LMSs) have shown great promise in cardiac research, allowing multicellular and complex interplay analyses with disease and patient specificity, yet their wider clinical use is limited by the large tissue sizes usually required. We therefore produced mini-LMSs (<10 mm²) from routine human cardiac surgery specimens and compared them with medium (10-30 mm²) and large (>30 mm²) slices. Size effects on biomechanical properties were examined with mathematical modeling, and viability, contraction profiles, and histological integrity were followed for 14 days. In total, 34 mini-, 25 medium, and 30 large LMS were maintained viable, the smallest measuring only 2 mm². Peak twitch force proved to be size-independent, whereas time-to-peak shortened as slice area decreased. Downsized LMSs displayed excellent contractile behavior for five to six days, after which a gradual functional decline and micro-architectural changes emerged. These findings confirm, for the first time, that mini-LMSs are feasible and viable, enabling short-term, patient-specific functional studies and pharmacological testing when tissue is scarce.

Keywords: 3D culture systems; experimental methods; in vitro models; living myocardial slices (LMSs); translational cardiology.

Figure 1

Mini-LMS preparation. (a) A representative sample of ventricular tissue obtained from a patient. (b) Ventricular tissue block embedded in agarose to ensure stabilization during slicing. (c) Tissue block mounted on a vibratome, ready for precise sectioning. (d) Triangular mounting devices (TMDs) used for holding and stabilizing the LMSs during cultivation. (e) Photographic record showcasing the orientation, size, and condition of the tissue slice post-sectioning. (f) A glimpse into the biomimetic cultivation chambers (BMCCs) housing the LMSs for their designated culture period.

Figure 2

Baseline biomechanical profiles of mini-, medium and large LMSs. (a) Bell curve with the biomechanical characteristics of contractility: AUC = area under the curve. CD = total contraction duration. F_max = maximum contraction force. TTP = time-to-peak. TTR = time-to-relaxation. +dF/dt = steepest positive slope. −dF/dt = steepest negative slope. (b) Three contraction curves of nonlinear regression mean values for large, medium, and mini-LMSs (the colors represent large (red), medium (green), and mini-LMSs (blue)). (c) Radar plot of median values, specified by LMS size category (the colors represent the same categories as in (b)).

Figure 3

Continuous contractility recording. Positive spikes of contractility were produced by isoprenaline administration and corresponded to medium exchange intervals (36–48 h). (a) Contractility from mini-LMSs from two different patients. (b) Contractility from large-LMSs from first patient.

Figure 4

Histology analysis of cultured slices. (a) Myocardial tissue architecture after long-term culture (2 weeks) was monitored by hematoxylin and eosin (H & E) staining. (b) Sirius Red staining was performed for tissue fibrosis (indicated in black arrow). (c) Quantification of Sirius Red intensity showed increased fibrosis at days 5, 10, and 14 compared to baseline (d0). Representative image shown (n = 1). (d) Immunostaining analysis of α-SMA (green) and α-ACTININ (red) revealed increasing myocardial fibrosis in cultured slices (highlighted in white). (e) Quantification of mean α-SMA intensities in d0, d5, d10, and d14 showed elevated levels of α-SMA in d10 and d14 slices compared to baseline (d0). Data represent mean ± SD from five slices per timepoint (n = 5). Abbreviations: H & E—hematoxylin and eosin; α-SMA—alpha-smooth muscle actin; α-ACTININ—sarcomeric alpha-Actinin. Statistics: Data in (c,e) were analyzed using two-tailed unpaired t-tests comparing each timepoint to d0. Asterisks indicate significance: p < 0.01 (**); p < 0.0001 (****).

Figure 5

Immunostaining analysis of cultured slices (a) CX43/GJA1 (green), α-ACTININ (red), and Hoechst 33342 (blue) staining was performed to visualize proteins at intercalated discs (ICDs), striated sarcomere, and nuclei of myocardial cells. (b) Quantification of signal intensity measurements of CX43/GJA1 at ICDs showing decreasing intensities in slices cultured longer, i.e., d5, d10, and d14, when compared to baseline (d0). Data represent mean ± SD from five slices per timepoint (n = 5). (c) Immunostaining analysis of α-Actin (green), showing irregular α-Actin stainings in d5, d10, and d14 when compared to baseline (d0). α-Actin was also seen at ICD in d5 slice (indicated in red arrow). (d) Quantification of cell length in d0, d5, d10, and d14 using CX43 staining (green) in A as cell boundaries revealed decreasing cell length in d5, d10, and d14 slices compared to baseline (d0). Data represent mean ± SD from five slices per timepoint (n = 5). Abbreviations: CX43—Connexin 43; α-ACTININ—sarcomeric alpha-Actinin; ICDs—intercalated discs; α-ACTIN—alpha Actin. Statistics: Data in (b,d) were analyzed using two-tailed unpaired t-tests comparing each timepoint to d0. Asterisks indicate significance: p < 0.01 (**); p < 0.001 (***).