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.
Tenaya Therapeutics, South San Francisco, CA (Z.J., K.N.I.).
Circ Res. 2019 Aug 30;125(6):628-642. doi: 10.1161/CIRCRESAHA.119.314996. Epub 2019 Jul 16.
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.
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.
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.
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.
在临床前阶段，新型心力衰竭治疗方法的心脏毒性和疗效测试面临一个主要限制：缺乏模拟人类心脏组织复杂性并能长时间保持活力和功能的原位培养系统。
开发一种可靠、易于复制的高通量方法，在生理条件下长时间培养猪和人心切片。
本研究描述了一种新颖的高通量仿生培养系统，该系统可在培养条件下维持人心切片（300μm 厚）和猪心切片长达 6 天的活力和功能。通过以 1.2 Hz 的频率进行连续电刺激和对培养基进行氧合，我们优化了培养基和培养条件。通过评估钙稳态、抽搐力产生和对β-肾上腺素能刺激的反应，证实这些切片在 6 天内具有功能活力。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，在长达 6 天的时间内，总体上维持了正常的基因表达，而在 10 天后，有超过 500 个转录本发生差异调节。电子显微镜显示，在优化条件下培养 6 天后，线粒体和 Z 盘超微结构完整。这种仿生培养系统成功地使人心切片在培养 6 天内保持完全存活、功能和结构完整。我们还使用该系统在人心切片中证明了一种新型基因治疗方法的效果。此外，该培养系统还使我们能够在培养后评估人心切片中分离的单个肌原纤维的收缩和松弛动力学。
我们已经开发并优化了一种可靠的高通量猪和人心切片培养系统，作为测试新型心力衰竭治疗方法疗效和在 3 维心脏模型中可靠测试心脏毒性的平台。
