title: The impact of left ventricular stretching in model cultivations with neonatal cardiomyocytes in a whole-heart bioreactor.
abstract: Here, we investigate the impact of integrated three-dimensional (3D) left ventricular (LV) stretching on myocardial maturation in a whole-heart bioreactor setting. Therefore, decellularized rat hearts were selectively repopulated with rodent neonatal cardiomyocytes (5 · 106cells per heart) and cultured over 5 days. Continuous medium perfusion was maintained through the coronary artery system in a customized whole-heart bioreactor system with or without integrated biomechanical stimulation of LV. 3D repopulation effectiveness and cellular vitality were evaluated by repetitive metabolic WST-1 assays and 3D confocal microscopy analysis through fluorescent staining, also assessing cellular organization. Moreover, specific myocardial vitality was verified by detecting spontaneous electrophysiological activity using a multielectrode assay. Western blot analysis of cardiac myosin heavychain (MHC) and quantitative RT-PCR for Connexin 43 was used to analyze cardiomyocyte maturation. Decellularized whole-heart constructs repopulated with neonatal cardiomyocytes (repopWHC) showed vital 3D cell populations throughout the repopulation sites within the LV with a significant increase in metabolic activity (326 ± 113% for stimulated constructs vs. 162 ± 32% for non-stimulated controls after 96 h of continuous cultivation as compared to their state 24 h after injection, directly prior to bioreactor cultivation). Further, bioreactor cultivation under integrated mechanical LV stimulation not only led to a higher degree of cellular organization and an increased MHC content, but also to a significant increase of Cx43 gene expression resulting in a regain of 60 ± 19% of native neonatal hearts expression level in contrast to 20 ± 9% for non-stimulated controls (P = 0.03). Therefore, our study suggests that the integration of LV stretching into whole-heart bioreactor cultivation may enhance cardiac maturation not only by promoting cellular organization but also through adaptive protein and gene expression with particular implications for the formation of the conductive apparatus. Further, this study emphasizes the importance of suitable bioprocessing strategies within sophisticated bioreactor systems as tools for customized stimulation and cultivation of tissue engineered tissues and organs. Biotechnol. Bioeng. 2017;114: 1107-1117. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Medical Faculty, Research Group for Experimental Surgery, Department of Cardiovascular Surgery, Heinrich Heine University, Moorenstr. 5, Duesseldorf, North Rhine-Westphalia, Germany, 40225.
authors.forename: Jörn
authors.initials: J
authors.lastname: Hülsmann
authors.affiliation: Medical Faculty, Research Group for Experimental Surgery, Department of Cardiovascular Surgery, Heinrich Heine University, Moorenstr. 5, Duesseldorf, North Rhine-Westphalia, Germany, 40225.
authors.forename: Hug
authors.initials: H
authors.lastname: Aubin
authors.affiliation: Medical Faculty, Research Group for Experimental Surgery, Department of Cardiovascular Surgery, Heinrich Heine University, Moorenstr. 5, Duesseldorf, North Rhine-Westphalia, Germany, 40225.
authors.forename: Alexander
authors.initials: A
authors.lastname: Wehrmann
authors.affiliation: Medical Faculty, Research Group for Experimental Surgery, Department of Cardiovascular Surgery, Heinrich Heine University, Moorenstr. 5, Duesseldorf, North Rhine-Westphalia, Germany, 40225.
authors.forename: Artur
authors.initials: A
authors.lastname: Lichtenberg
authors.affiliation: Medical Faculty, Research Group for Experimental Surgery, Department of Cardiovascular Surgery, Heinrich Heine University, Moorenstr. 5, Duesseldorf, North Rhine-Westphalia, Germany, 40225.
authors.forename: Payam
authors.initials: P
authors.lastname: Akhyari
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001696:Biomechanical Phenomena / Q000502:physiology*; D019149:Bioreactors; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D008297:Male; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D023822:Tissue Engineering / Q000379:methods*; D016276:Ventricular Function / Q000502:physiology*
keywords: biomechanical stimualtion; bioprocessing systems; left-ventricular strechting; myocardial tissue engineering; whole-heart bioreactor