Summary

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Published: June 16, 2020
doi:

Summary

该协议描述了用于临床前药物测试的人体心脏切片切片和培养过程,并详细介绍了光学映射用于同时记录这些切片的跨膜电压和细胞内钙信号的过程。

Abstract

人类心脏切片制剂最近被开发为人类生理学研究和治疗测试的平台,以弥合动物和临床试验之间的差距。许多动物和细胞模型被用来检查药物的作用,但这些反应在人类中往往不同。人类心脏切片为药物测试提供了一个优势,因为它们直接来自可行的人类心脏。除了保留了多细胞结构、细胞耦合和细胞外基质环境外,人类心脏组织切片还可用于直接测试无数药物对成人心脏生理学的影响。这种模式与其他心脏制剂(如整个心脏或楔形)的区别,是切片可以受长期文化的影响。因此,心脏切片允许研究药物的急性和慢性影响。此外,从一颗心脏收集几百到一千片切片的能力使得这是一种高通量模型,可以同时测试不同浓度和与其他药物组合的几种药物。切片可以从心脏的任何给定区域制备。在此协议中,我们描述了左心室切片的制备,通过将组织立方体从左心室自由壁中分离,并使用高精度振动微孔将它们分割成切片。然后,这些切片可以进行急性实验,以测量基线心脏电生理功能,或培养用于慢性药物研究。该协议还描述了心脏切片的双光学映射,用于同时记录跨膜电位和细胞内钙动力学,以确定被调查药物的影响。

Introduction

动物模型是了解人类生理学和病理生理学基本机制的宝贵工具,也是初步测试治疗各种疾病疗法的平台。基于这些动物研究的生物医学研究领域取得了长足的进步。然而,人类和动物生理之间存在着显著的物种间差异,包括小鼠、大鼠、豚鼠、兔子、绵羊、猪和狗33、4。4因此,在动物试验阶段,有许多药物、基因和细胞疗法显示出希望,但未能达到临床试验结果5。为了弥补这一差距,分离的心肌细胞和人类诱导多能干细胞(iPSCs)被开发为模型,以测试人类生理对各种药物和疾病的反应6。干细胞衍生心肌细胞作为心脏66、7、87,8的代用品,已广泛应用于芯片器官系统。然而,iPSC衍生的心肌细胞(iPSC-CMs)的效用因其相对不成熟的表型和心肌细胞亚群的代表性而受到阻碍;成熟的心肌是一种复杂的结构,由多种共存的细胞类型组成,如成纤维细胞、神经元、巨噬细胞和内皮细胞。另一方面,分离的人类心肌细胞在电成熟,通过改变培养参数9,可以获得不同的心肌细胞亚群。然而,由于缺乏细胞-细胞耦合、快速去分化以及体外10、11,11内出现多节律行为,这些肌细胞通常表现出改变的动作潜力形态。iPSC-CM 和心形肌细胞的 3D 细胞培养模型解决了一些限制。这些模型包括球菌、水凝胶支架封装的 3D 培养物、工程心脏组织 (EHT) 和片上心脏系统,使用多个心脏细胞群,如心肌细胞、成纤维细胞和内皮细胞。它们要么自组装,要么沿着脚手架组装形成3D结构,有些甚至重现了心肌的复杂各向异性。据报道,这些模型具有与心脏组织相似的成熟表型、收缩特性和分子轮廓的细胞。片上心脏系统还允许研究药物测试和疾病模型中的系统效应。然而,体外细胞基模型缺乏原生细胞外基质,因此不能准确模拟器官水平电生理学。相比之下,人类心脏切片具有完整的细胞外基质和原生细胞对细胞的接触,因此有助于更准确地检查人类心肌的心律失常特性。

研究人员已开发出人心形器官切片,作为急性和慢性药物测试的生理前临床平台,并研究心脏电生理学和心脏病进展12、13、14、15、16、17、18、19。12,13,14,15,16,17,18,19与 iPSC 衍生的心肌细胞相比,人类心脏切片更忠实地复制了成熟的心肌细胞表型成人心脏电生理学。与分离的人类心肌细胞相比,心脏切片表现出生理作用潜力持续时间,因为细胞耦合保存完好,并且其原生细胞内和细胞外环境存在。

该协议描述了从整个供体心脏生成人类心脏切片的过程,进行急性(即数小时)和慢性(即数天)研究,通过光学映射测试心脏电生理学参数的过程。虽然该协议只描述了左心室(LV)组织的使用,但它已成功应用于心脏的其他地区以及其他物种,如小鼠、大鼠、豚鼠和猪14、20、21、22。14,20,21,22我们的实验室使用过去5年被拒绝移植的全身供体心脏,但只要组织能够分割成立方体,这些相同的程序可以用于通过其他手段获得的任何供体心脏样本组织(例如左心室辅助装置[LVAD]植入、活检、骨髓切除术)。光学映射用于本研究的分析,因为光学映射具有高空间(100 x 100 像素)和时间(>1,000 帧/秒)分辨率的光学动作电位和钙瞬变。也可以使用替代方法,例如多电极阵列 (MEA) 或微电极,但这些技术受到空间分辨率相对较低的限制。此外,MEA 被设计为用于细胞培养物,锋利的微电极更易于管理,用于整个心脏或大型组织楔块。

本文的目标是让更多的研究人员使用人类心脏组织进行心脏电生理学研究。需要注意的是,本文中描述的技术相对简单,对短期研究(按几个小时到几天的顺序)有益。其他一些研究12、18、23,18,23已经讨论和描述了用于长期研究的更多生理生物仿生培养(周数)。电刺激、机械加载和组织拉伸是有利的调节机制,可以帮助限制体外组织重塑12、18、2318,2312发病。

Protocol

所述的所有方法都符合所有机构、国家和国际人类福利准则。研究得到了乔治华盛顿大学机构审查委员会(IRB)的批准。 注:经乔治华盛顿大学IRB批准,从华盛顿地区移植社区获得捐赠人的心脏作为被解认的废弃组织。通过用冰冷心性心肌痛溶液冲洗心脏(在此过程中血液从心脏被清除),并在标准器官移植条件下转移到实验室,使心脏被心肺复苏。 1. ?…

Representative Results

根据上面详述的规程,从供体人类心脏的左心室收集人体器官切片,并图中所示图 1.双摄像头光学映射系统,如图 2用于直立成像配置,在切片协议完成后约 1 小时对电压和钙进行同步光学映射。使用 RHYTHM1.2 对数据进行分析(图 3),一个开源光学映射数据分析工具,以前由我们的实验室发布,可在 Github (https://github.com/optocardio…

Discussion

在这里,我们提出了分步的方法,从心肌梗活的人类心脏获得可行的心脏切片,并使用跨膜电位和细胞内钙的双重光学映射功能地描述切片。通过保存的细胞外环境和原生细胞耦合,人类心脏切片可用作人类心脏的准确模型,用于基本科学发现,以及药理剂和基因疗法的疗效和心毒性测试。该技术还允许对心脏的特定区域进行结构功能映射,如心体节点、三角结和Purkinje纤维。此处描述的协议列?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

由NIH资助(资助R21 EB023106,R44 HL139248和R01 HL126802),由利杜克基金会(项目RHYTHM)和美国心脏协会博士后奖学金(19POST34370122)感谢。

Materials

1mL BD Syringe Thomas Scientific 309597
2,3-butanedione monoxime Sigma-Aldrich B0753
6 well culture plates Corning 3516
Biosafety cabinet ThermoFisher Scientific 1377
Blebbistatin Cayman 13186
Bubble Trap Radnoti 130149
Calcium chloride Sigma-Aldrich C1016
Corning Cell Strainers Fisher Scientific 07-201-432
Di-4-ANEPPS Biotium stock solution at 1.25 mg/mL in DMSO
DMSO Sigma-Aldrich D2650
Dumont #3c Forceps Fine Science Tools 11231-20
Emission dichroic mirror Chroma T630LPXR-UF1
Emission filter (RH237) Chroma ET690/50m
Emission Filter (Rhod2AM) Chroma ET590/33m
Excitation dichroic mirror Chroma T550LPXR-UF1
Excitation Filter Chroma ET500/40x
Falcon 50mL Conical Centrifuge Tubes Fisher Scientific 14-959-49A
Glucose Sigma-Aldrich G8270
Heat exchanger Radnoti 158821
HEPES Sigma-Aldrich H3375
Incubator ThermoFisher Scientific 50145502
Insulin Transferrin Selenium (ITS) Sigma-Aldrich I3146
LED excitation light source Prizmatix UHP-Mic-LED-520
Magnessium chloride hexahydrate Sigma-Aldrich M9272
Medium 199 ThermoFisher Scientific 11150059
Micam Ultima L type CMOS camera Scimedia N/A
Minutien Pins Fine Science Tools 26002-10
Pennicillin-Streptomycin Sigma-Aldrich P4333
Peristaltic Pump Cole Parmer EW-07522-20
Platinum pacing wire Alfa Aesar 43275
Pluronic F127 ThermoFisher Scientific P6867 nonionic, surfactant polyol
Potassium chloride Sigma-Aldrich P3911
Powerlab data acquisition and stimulator AD Instruments Powerlab 4/26
RH237 Biotium 61018
Rhod2AM ThermoFisher Scientific R1245MP
Rhod-2AM Invitrogen, Carlsbad, CA
Sodium bicarbonate Sigma-Aldrich S6014
Sodium chloride Sigma-Aldrich S9625
Sterilizer, dry bead Sigma-Aldrich Z378550
Stone Oxygen Diffuser Waterwood B00O0NUVM0
TissueSeal – Histoacryl Topical Skin Adhesive gobiomed AESCULAP
UltraPure Low Melting Point Agarose Thermo Fisher Scientific 16520100
Ultrasound sonicator Branson 1800
Vibratome Campden Instruments 7000 smz

References

  1. Ericsson, A. C., Crim, M. J., Franklin, C. L. A brief history of animal modeling. Missouri Medicine. 110 (3), 201-205 (2013).
  2. Choudhary, A., Ibdah, J. A. Animal models in today’s translational medicine world. Missouri Medicine. 110 (3), 220-222 (2013).
  3. Perlman, R. L. Mouse models of human disease: An evolutionary perspective. Evolution, Medicine, and Public Health. 2016 (1), 170-176 (2016).
  4. Milani-Nejad, N., Janssen, P. M. L. Small and large animal models in cardiac contraction research: advantages and disadvantages. Pharmacology & Therapeutics. 141 (3), 235-249 (2014).
  5. Green, A. R. Why do neuroprotective drugs that are so promising in animals fail in the clinic? An industry perspective. Clinical and Experimental Pharmacology and Physiology. 29 (11), 1030-1034 (2002).
  6. Shinnawi, R., Gepstein, L. iPCS cell modeling of inherited cardiac arrhythmias. Current Treatment Options in Cardiovascular Medicine. 16 (9), 331 (2014).
  7. Morimoto, Y., Mori, S., Sakai, F., Takeuchi, S. Human induced pluripotent stem cell-derived fiber-shaped cardiac tissue on a chip. Lab on a Chip. 16 (12), 2295-2301 (2016).
  8. Wang, G., et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nature Medicine. 20 (6), 616-623 (2014).
  9. Ben-Ari, M., et al. Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes. Heart Rhythm. 13 (12), 2379-2387 (2016).
  10. Goversen, B., van der Heyden, M. A. G., van Veen, T. A. B., de Boer, T. P. The immature electrophysiological phenotype of iPSC-CMs still hampers in vitro drug screening: Special focus on IK1. Pharmacology & Therapeutics. 183, 127-136 (2018).
  11. Zhang, Y., et al. Dedifferentiation and proliferation of mammalian cardiomyocytes. PloS One. 5 (9), 12559 (2010).
  12. Watson, S. A., et al. Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro. Nature Communications. 10, 2168 (2019).
  13. Fischer, C., et al. Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro. Nature Communications. 10, 117 (2019).
  14. Ou, Q., et al. Physiological Biomimetic Culture System for Pig and Human Heart Slices. Circulation Research. 125 (6), 628-642 (2019).
  15. Qiao, Y., et al. Multiparametric slice culture platform for the investigation of human cardiac tissue physiology. Progress in Biophysics and Molecular Biology 2. 144, 139-150 (2019).
  16. Kang, C., et al. Human Organotypic Cultured Cardiac Slices: New Platform For High Throughput Preclinical Human Trials. Scientific Reports. 6, 28798 (2016).
  17. Camelliti, P., et al. Adult human heart slices are a multicellular system suitable for electrophysiological and pharmacological studies. Journal of Molecular and Cellular Cardiology. 51 (3), 390-398 (2011).
  18. Brandenburger, M., et al. Organotypic slice culture from human adult ventricular myocardium. Cardiovascular Research. 93 (1), 50-59 (2012).
  19. Watson, S. A., et al. Preparation of viable adult ventricular myocardial slices from large and small mammals. Nature Protocols. 12 (12), 2623-2639 (2017).
  20. Halbach, M., et al. Ventricular slices of adult mouse hearts – A new multicellular in vitro model for electrophysiological studies. Cellular Physiology and Biochemistry. 18 (1-3), 1-8 (2006).
  21. Wang, K., et al. Cardiac tissue slices: preparation, handling, and successful optical mapping. American Journal of Physiology. Heart and Circulatory Physiology. 308 (9), 1112-1125 (2015).
  22. Bussek, A., et al. Cardiac tissue slices with prolonged survival for in vitro drug safety screening. Journal of Pharmacological and Toxicological Methods. 66 (2), 145-151 (2012).
  23. Watson, S. A., Terracciano, C. M., Perbellini, F. Myocardial Slices: an Intermediate Complexity Platform for Translational Cardiovascular Research. Cardiovascular Drugs and Therapy. 33 (2), 239-244 (2019).
  24. Rouwkema, J., Koopman, B. F. J. M., Blitterswijk, C. A. V., Dhert, W. J. A., Malda, J. Supply of nutrients to cells in engineered tissues. Biotechnology and Genetic Engineering Reviews. 26 (1), 163-178 (2009).
  25. Lang, D., Sulkin, M., Lou, Q., Efimov, I. R. Optical mapping of action potentials and calcium transients in the mouse heart. Journal of Visualized Experiments. (55), e3275 (2011).
  26. Brianna, C., et al. Open-Source Multiparametric Optocardiography. Scientific Reports. 9, 721 (2019).
  27. George, S. A., et al. Modulating cardiac conduction during metabolic ischemia with perfusate sodium and calcium in guinea pig hearts. American Journal of Physiology – Heart and Circulatory Physiology. 316 (4), 849-861 (2019).
  28. Kawara, T., et al. Activation delay after premature stimulation in chronically diseased human myocardium relates to the architecture of interstitial fibrosis. Circulation. 104 (25), 3069-3075 (2001).
  29. Qu, Y., et al. Action potential recording and pro-arrhythmia risk analysis in human ventricular trabeculae. Frontiers in Physiology. 5 (8), 1109 (2018).
  30. Franz, M. R., Swerdlow, C. D., Liem, L. B., Schaefer, J. Cycle length dependence of human action potential duration in vivo. Effects of single extrastimuli, sudden sustained rate acceleration and deceleration, and different steady-state frequencies. Journal of Clinical Investigation. 82 (3), 972-979 (1988).
  31. Lou, Q., et al. Transmural heterogeneity and remodeling of ventricular excitation- contraction coupling in human heart failure. Circulation. 123 (17), 1881-1890 (2011).

Play Video

Cite This Article
George, S. A., Brennan, J. A., Efimov, I. R. Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices. J. Vis. Exp. (160), e60781, doi:10.3791/60781 (2020).

View Video