Physiological Biomimetic Culture System for Pig and Human Heart Slices
Physiological Biomimetic Culture System for Pig and Human Heart Slices
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DOI：
10.1161/circresaha.119.314996
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发表时间：
2019-08-30
影响因子：
20.1
通讯作者：
Mohamed, Tamer M. A.
中科院分区：
文献类型：
--
作者：
Ou, Qinghui;Jacobson, Zoe;Mohamed, Tamer M. A.
基本原理：新型心力衰竭疗法的心脏毒性和疗效的临床前测试面临着一个主要的限制：缺乏模拟人类心脏组织复杂性并长时间保持活力和功能的原位培养系统。目的：建立一种可靠、易重复、中等通量的方法，在生理条件下长时间培养猪和人的心脏切片。方法与结果：在这里，我们描述了一种新的，中等吞吐量的仿生培养系统，保持活力和功能的人和猪的心脏切片（300 μ m厚）6天的文化。我们优化了培养基和培养条件，以1.2 Hz连续电刺激和培养基的充氧。通过评估这些切片的钙稳态、抽搐力产生和对β-肾上腺素能刺激的反应，证实了这些切片在6天内的功能活力。在培养的第2、6和10天使用RNAseq进行的时间转录组分析证实了正常基因表达的总体维持长达6天，而超过500个转录本在10天后被差异调节。电子显微镜显示，在我们优化的条件下培养6天后，完整的线粒体和Z盘超微结构。这种仿生培养系统成功地使人心脏切片在培养中完全存活，功能和结构完整6天。我们还使用该系统来证明一种新的基因治疗方法在人类心脏切片中的效果。此外，该培养系统能够评估培养后来自心脏切片的分离的单肌原纤维的收缩和舒张动力学。结论：我们已经开发并优化了一个可靠的猪和人心脏切片的中通量培养系统，作为一个平台，用于测试新型心力衰竭治疗剂的疗效和在三维心脏模型中可靠的心脏毒性测试。可视化概述：本文提供了一个在线可视化概述。
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 mu m 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 beta-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. Visual Overview: An online visual overview is available for this article.