Qinghui Ou1, Zoë Jacobson2, Riham R E Abouleisa1, Xian-Liang Tang1, Sajedah M Hindi3, Ashok Kumar3, Kathryn N Ivey2, Guruprasad Giridharan4, Ayman El-Baz4, Kenneth Brittian1, Benjamin Rood5, Ying-Hsi Lin6, Samuel A Watson7, Filippo Perbellini7,8, Timothy A McKinsey6, Bradford G Hill5, Steven P Jones1,5, Cesare M Terracciano7, Roberto Bolli1, Tamer M A Mohamed1,5,9,10,11. 1. From the Department of Medicine, Institute of Molecular Cardiology (Q.O., R.R.E.A., X.-L.T., K.B., S.P.J., R.B., T.M.A.M.), University of Louisville, KY. 2. Tenaya Therapeutics, South San Francisco, CA (Z.J., K.N.I.). 3. Departments of Anatomical Sciences and Neurobiology (S.M.H., A.K.), University of Louisville, KY. 4. Department of Bioengineering (G.G., A.E.-B.), University of Louisville, KY. 5. Envirome Institute, Diabetes and Obesity Center, Department of Medicine (B.R., B.G.H., S.P.J., T.M.A.M.), University of Louisville, KY. 6. Division of Cardiology and Consortium for Fibrosis Research & Translation, Department of Medicine, University of Colorado, Aurora (Y.-H.L., T.A.M.). 7. National Heart & Lung Institute, Imperial College London, United Kingdom (S.A.W., F.P., C.M.T.). 8. Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Germany (F.P.). 9. Department of Pharmacology and Toxicology (T.M.A.M.), University of Louisville, KY. 10. Institute of Cardiovascular Sciences, University of Manchester, United Kingdom (T.M.A.M.). 11. Faculty of Pharmacy, Zagazig University, Egypt (T.M.A.M.).
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 β-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 3-dimensional heart model.
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 β-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 3-dimensional heart model.
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