Physiological Biomimetic Culture System for Pig and Human Heart Slices

Abstract

Rationale: Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. Objective: To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. Methods and Results: Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to -adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. Conclusions: We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3D heart model.

Minimally invasive system to reliably characterize ventricular electrophysiology from living donors

“…Upon vibratome cutting, the presence of damaged cells in the tissue slices is mainly restricted to the first outer layers of cells transected during the slicing, which nevertheless presents some viability. Cardiomyocytes' viability increases deep into the tissue slice, in agreement with previous studies on larger ventricular myocardial slices 7 , 11 , 12 , 18 , 20 , 33 .…”

“…The alignment of the myocardial fibers with the vibratome slicing plane improves viability and restricts tissue damage to the very first cellular layers, as previously reported 6 , 13 , 15 , 16 , 18 , 33 . The fiber alignment step is relevant not only to transmural core biopsies, but generally to any other tissue to be sliced.…”

Epicardial slices: a 3D organotypic model for the study of epicardial activation and differentiation

“…Epicardial slices maintain normal heart tissue architecture, as demonstrated by the preservation of functional markers and architecture in the myocardial portion of the slice, comparable with previous reports in myocardial slices (19,29,30). More importantly, slices present an intact epicardial monolayer expressing typical markers such as MSLN, E-cadherin and WT1.…”

“…Our results show that both culture systems preserve an unaltered cobblestone-shaped epicardial layer and sustain the overall slice viability and metabolic rate, comparable to previously reported myocardial slices from large mammals (30,36). Notably, the more stable environment provided by the dynamic culture system promote proliferation of epicardial cells.…”

Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine

“…These limitations demonstrate the fundamental importance of the in vivo environment for maintaining the mature cardiac phenotype and highlight the shortcomings of standard cell culture techniques. Furthermore, these limitations have led to a re-exploration of the use of human adult heart tissue slices in long-term culture, which demonstrate impressive maintenance of structure and function and show promise for disease modelling and for toxicology and drug screening [225][226][227][228][229] . However, these models are currently limited by their fairly low throughput, the technical expertise required, and the inability to control for genetic diversity between individuals or perform and trace the developmental consequences of perturbations (for example, genome editing) compared with PSC-CMs.…”