Hülsmann Jörn, Aubin Hug, Wehrmann Alexander, Lichtenberg Artur, Akhyari Payam
Medical Faculty, Research Group for Experimental Surgery, Department of Cardiovascular Surgery, Heinrich Heine University, Moorenstr. 5, Duesseldorf, North Rhine-Westphalia, Germany, 40225.
Biotechnol Bioeng. 2017 May;114(5):1107-1117. doi: 10.1002/bit.26241. Epub 2017 Jan 11.
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 · 10 cells 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.
在此，我们在全心脏生物反应器环境中研究了整合的三维（3D）左心室（LV）拉伸对心肌成熟的影响。因此，用啮齿类动物新生心肌细胞（每颗心脏5×10个细胞）对脱细胞大鼠心脏进行选择性再填充，并培养5天。在定制的全心脏生物反应器系统中，通过冠状动脉系统维持连续的培养基灌注，该系统有或没有对左心室的整合生物力学刺激。通过重复代谢WST-1测定和荧光染色的3D共聚焦显微镜分析评估3D再填充效果和细胞活力，同时也评估细胞组织。此外，使用多电极测定法检测自发电生理活性来验证特定的心肌活力。通过心脏肌球蛋白重链（MHC）的蛋白质印迹分析和连接蛋白43的定量逆转录-聚合酶链反应来分析心肌细胞成熟。用新生心肌细胞再填充的脱细胞全心脏构建体（repopWHC）在左心室内的整个再填充部位显示出有活力的3D细胞群，代谢活性显著增加（连续培养96小时后，受刺激构建体为326±113%，未受刺激对照为162±32%，与注射后24小时即生物反应器培养前的状态相比）。此外，在整合机械左心室刺激下的生物反应器培养不仅导致更高程度的细胞组织和MHC含量增加，而且还导致连接蛋白43基因表达显著增加，与未受刺激对照的20±9%相比，恢复到天然新生心脏表达水平的60±19%（P = 0.03）。因此，我们的研究表明，将左心室拉伸整合到全心脏生物反应器培养中不仅可以通过促进细胞组织，而且还可以通过适应性蛋白质和基因表达来增强心脏成熟，这对传导系统的形成具有特别重要的意义。此外，本研究强调了在复杂的生物反应器系统中采用合适的生物加工策略作为定制刺激和培养组织工程组织和器官的工具的重要性。《生物技术与生物工程》2017年；114: 1107 - 1117。© 2016威利期刊公司
