Comparison of Culture Systems for Mouse Living Myocardial Slices in Cardiac Fibrosis Research

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Abstract Living myocardial slices (LMS) are a highly relevant ex vivo model for investigating cardiac physiology and disease, as they preserve the native three-dimensional architecture, cellular diversity, and extracellular matrix of the heart. In addition, LMS enable longitudinal functional and molecular analyses. In this study, we established and compared two LMS culture approaches: an air-liquid interface system and a biomimetic culture system. We further examined how different slicing techniques affect tissue quality and longevity within the biomimetic setup. To develop a fibrosis model, LMS were stimulated with transforming growth factor-β1 (TGF-β1) and/or exposed to increased mechanical load. Tissue viability was assessed using LIVE/DEAD staining and the MTT assay, while cytotoxicity was evaluated with the LDH-Glo™ Cytotoxicity assay. Contractile function was measured, and fibrotic remodelling was analysed using RT-qPCR, ELISA, and immunohistochemistry. Our results demonstrate that LMS cultured in the biomimetic system exhibit superior viability, structural integrity, and functional performance compared with those maintained at the air-liquid interface. Mouse LMS could be stably cultured for up to one week in the biomimetic system. Importantly, sample preparation, particularly the slicing method, had a significant impact on tissue quality and culture duration. While TGF-β1 stimulation alone did not consistently induce fibrosis, combining TGF-β1 treatment with increased mechanical load led to more pronounced fibrotic remodelling in LMS. These findings highlight the importance of biomechanical cues in modelling cardiac fibrosis ex vivo and support the biomimetic system as a robust platform for functional and disease-relevant studies. Highlights Biomimetic culture preserves viability, structure, and contractile function of mouse LMS better than air-quid interface culture. Sample preparation, specifically the slicing technique, critically determines LMS quality and culture longevity. Mouse LMS can be maintained for up to six days in a biomimetic system, enabling longitudinal functional analyses. Increased mechanical load and culture duration, not TGF-β1, drove fibrotic changes in LMS. Graphical abstract

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