Heart Center, University of Amsterdam, Amsterdam, The Netherlands.
PLoS One. 2013;8(3):e59290. doi: 10.1371/journal.pone.0059290. Epub 2013 Mar 13.
Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, -80.5±3.5 mV in freshly isolated tissue, and -60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy.
在体外测试心脏基因和细胞疗法需要一种组织底物，该底物在培养中能存活数天，同时保持其生理特性。本研究旨在测试完整的新生大鼠心室心脏组织（器官外植体培养）的培养是否可用于研究基因和细胞治疗的模型。我们将器官外植体培养物的（免疫）组织学和电生理学与新鲜分离的新生大鼠心室组织和单层细胞进行了比较。（免疫）组织学研究表明，器官外植体培养物保留了其纤维取向，并且α-肌动蛋白、连接蛋白 43 和α-平滑肌肌动蛋白的表达模式在培养过程中没有改变。细胞内电压记录显示，自发搏动在器官外植体培养物（20%）和新鲜分离的组织（17%）中很少见，但在单层细胞中很常见（82%）。因此，器官外植体培养物的静息膜电位为-83.9±4.4 mV，新鲜分离的组织为-80.5±3.5 mV，单层细胞为-60.9±4.3 mV。通过光学映射测量的传导速度在器官外植体培养物中为 18.2±1.0 cm/s，在新鲜分离的组织中为 18.0±1.2 cm/s，在单层细胞中为 24.3±0.7 cm/s。我们发现器官外植体培养物与新鲜分离的组织之间的动作电位持续时间（APD）没有差异，而单层细胞的 APD 延长（APD 在 70%复极化时分别为 88.8±7.8、79.1±2.9 和 134.0±4.5 ms）。器官外植体培养物和新鲜分离的组织可以起搏到新生大鼠的正常范围内的频率（CL 150 ms），而单层细胞则不能。通过 Egfp 基因转移显示出成功的慢病毒（LV）转导。与自发搏动的心肌细胞共培养可使器官外植体培养物的自发搏动活动发生率增加到 86%。我们得出结论，新生大鼠心室的器官外植体培养物在结构和电生理学上与新鲜分离的组织相似，是研究基因和细胞治疗效果的合适新模型。
