JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Divulgaciones
Acknowledgements
Materials
Referencias
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Bioingeniería

在微流体平台内开发 3D 组织人类心脏组织

Published: June 15, 2021
doi:
10.3791/62539
,
1School of Biological and Health Systems Engineering,Arizona State University, 2Biodesign Virginia G. Piper Center for Personalized Diagnostics,Arizona State University

Summary

本协议的目的是解释和演示高度对齐的人类心脏组织的三维（3D）微流体模型的发展，该模型由干细胞衍生心肌细胞组成，与心脏成纤维细胞（CFs）在生物仿真、胶原蛋白基水凝胶中共同培养，用于心脏组织工程、药物筛查和疾病建模。

Abstract

全世界的主要死因是心血管疾病（CVD）。然而，建模心脏肌肉的生理和生物学复杂性，心肌，是出了名的难以 完成体外。 主要，障碍在于需要人类心肌细胞（CMs），这些细胞要么是成人的，要么是显示成人样的表型，并且能够成功地复制心肌的细胞复杂性和复杂的3D结构。不幸的是，由于伦理问题和缺乏可用的原发性患者衍生人类心脏组织，加上CS的微小增殖，可行的人类CMs的采购一直是心脏组织工程的一个限制步骤。为此，大多数研究已经过渡到人类诱导多能干细胞（hiPSC）作为人类CMs的主要来源的心脏分化，导致 在体外 测定中广泛纳入HIPSC-CMs，用于心脏组织建模。

在这项工作中，我们演示了在微流体设备中开发3D成熟干细胞衍生人类心脏组织的方案。我们特别解释和视觉上演示了从hiPSC衍生的CMs中产生的3D 体外 不运动心脏组织芯片模型的生产。我们主要描述为 CMs 选择的纯化协议，通过将 CMs 与人类 CF （hCF） 混合来共同培养具有定义比率的细胞，以及暂停胶原蛋白水凝胶中的这种共同培养。我们进一步演示了在我们定义明确的微流体装置中注入细胞载水凝胶，内嵌交错椭圆微柱，作为表面地形，诱导周围细胞和水凝胶基质的高度对齐，模仿原生心肌的结构。我们设想，拟议的3D非同向心脏组织芯片模型适用于基础生物学研究，疾病建模，并通过它作为筛选工具，药物测试。

Introduction

组织工程方法已广泛探索，近年来，伴随着体内临床发现再生医学和疾病建模1，2。由于在采购人类原发性心脏组织和产生生理相关体外代孕方面存在固有困难，因此特别强调体外心脏组织建模，这限制了对心血管疾病（CVDs）1、3复杂机制的基本理解。传统模型通常涉及 2D 单层文化测定。然而，在3D环境中培养心脏细胞，模仿心肌原生景观和复杂的细胞相互作用的重要性，已被广泛定性为4，5。此外，迄今为止生产的大多数模型都包括了与干细胞不同的CMs的单一培养。然而，心脏是由多个细胞类型6在复杂的3D架构7，保证迫切需要改善组织成分的复杂性在3D体外模型，以更好地模仿细胞成分的原生心肌。

迄今为止，已经探索了许多不同的方法来生产模因8的生物仿生3D模型。这些方法的范围从允许实时计算生成力的实验设置，从在薄膜（被认为是肌肉薄膜 （MTFs）9上播种的单培养 CMs，到悬浮在自立的钳子（被视为工程心脏组织 （EHTs）10中的 3D 水凝胶基质中的共同培养心脏细胞。其他方法则侧重于实施微模技术，以模仿心肌厌食症，从11号组织贴片中悬浮在突出微柱中的3D水凝胶中的单培养CMs，到在缩化微沟12、13中播种的单培养CMs。因此，利用与预期应用和相应的生物学问题相一致的技术，每种方法都有内在的优点和缺点。

增强干细胞衍生 CMs 成熟度的能力对于成功进行成人心肌组织体外工程以及将后续发现转化为临床解释至关重要。为此，在2D和3D14、15、16方面，对成熟CMs的方法进行了广泛的探索。例如，EHT中所包含的电刺激、CM与地表地形、信号提示、共同培养的生长因子和/或3D水凝胶条件等的强制对齐，都导致至少在以下一种情况下有利于CM成熟的变化：细胞形态、钙处理、肉瘤结构、基因表达或收缩力。

在这些模型中，利用微流体平台的方法在性质上保留了某些优势，例如控制梯度、有限的细胞输入和最少必要的试剂。此外，许多生物复制品可以立即产生使用微流体平台，有助于更好地解剖生物利益机制，增加实验样本量，有利于统计力17，18，19。此外，在微流体设备制造过程中使用光刻技术，在微观和纳米层面创建精确的特征（如地形图），作为中微线索，增强周围的细胞结构和宏观级组织结构18、20、21、22，用于组织再生和疾病建模的不同应用。

我们以前曾证明开发一种新的3D心脏组织片上模型，结合表面地形，以天生椭圆形微柱的形式，将水凝胶封装共同培养的心脏细胞对齐成一个相互连接的，不亚素组织20。经过14天的培养，与单层和3D同位素控制23相比，微流体装置内形成的组织在表型、基因表达特征、钙处理特性和药物反应等方面更加成熟。此处描述的协议概述了使用hiPSC衍生的 CMs 在微流体设备中创建这种 3D 共培养、对齐（即不运动）人类心脏组织的方法。 具体来说，我们解释区分和净化高浓缩丙基对CMs的方法，补充HCF与CMs产生一个既定的共同培养群体，将细胞群封装在胶原水凝胶中到微流体设备中，以及随后通过收缩和免疫荧光检测分析3D构造组织。由此产生的3D工程微组织适用于各种应用，包括基础生物学研究、CVD建模和药物测试。

Protocol

在生物安全柜内执行所有细胞处理和试剂准备。确保与细胞接触的所有表面、材料和设备均无菌（即喷洒70%乙醇）。细胞应在加湿的 37 °C、5% CO2 孵化器中培养。所有hiPSC文化和分化均在6井板中执行。 1. 微流体设备创建（大致持续时间：1 周） 光刻注：使用CAD文件设计的面膜（作为 补充文件1提供），包含微流体通道的设计。将设?…

Representative Results

为了从高PSC获得高度纯化的CMs群，使用了涉及连分化协议33和富山净化步骤34组合的修改版本（参照图1A表示实验时间线）。hiPSC 需要像殖民地一样，约 85% 的汇合，并在通过 3-4 天后，在 CM 分化（图 1B）的开始，均匀地传播到整个文化中。具体来说，在第 0 天，hiPSC 殖民地应具有多能转录因子的高表达性，包括 SOX2 ?…

Discussion

形成具有增强细胞相互作用和仿生3D结构的体外人体心脏组织模型，对于基本的心血管研究和相应的临床应用至关重要。此概述的协议解释了微流体设备中 3D 人类不运动性心脏组织的发展，使用干细胞衍生 CMs 与连接 CFs 共同培养，这些 CMs 封装在胶原蛋白水凝胶中，以模拟原生心肌的复杂细胞组成和结构。这个特定的协议是高度可重复的，因为设备的特殊结构已经优化和?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们要感谢NSF职业奖#1653193，亚利桑那州生物医学研究委员会（ABRC）新调查员奖（ADHS18-198872），以及弗林基金会奖为该项目提供资金来源。HIPSC线SCVI20来自由NIH R24 HL117756资助的斯坦福心血管研究所博士吴约瑟。HIPSC线，IMR90-4，是从威塞尔研究所55，56获得。

Materials

0.65 mL centrifuge tubes VWR 87003-290
1 mm Biopsy punch VWR 95039-090
1.5 mm Biopsy punch VWR 95039-088
15 mL Falcon tubes VWR 89039-670
18x18mm coverslips VWR 16004-308 The coverslips should be No.1, to allow for high magnification imaging
4% paraformaldehyde ThermoFisher 101176-014
6-well flat botttom tissue-culture plates VWR 82050-844
B27 minus insulin LifeTech A1895602
B27 plus insulin LifeTech 17504001
CHIR99021 VWR 10188-030
Collagen I, rat tail Corning 47747-218
DMEM F12 ThermoFisher 11330057
DPBS ThermoFisher 21600069
E8 ThermoFisher A1517001 can also be made in house
EDTA VWR 45001-122
Ethanol
FGM3 VWR 10172-048
GFR-Matrigel VWR 47743-718
Glycine Sigma G8898-500G
Goat serum VWR 10152-212
hESC-Matrigel Corning BD354277
IPA
IWP2 Sigma I0536-5MG
Kimwipes VWR 82003-820
MTCS Sigma 440299-1L
NaN3 Sigma S2002-25G
NaOH Sigma S5881-500G
Pen/Strep VWR 15140122
Petri dish (150x15mm) VWR 25384-326
Petri dish (60x15mm) VWR 25384-092
Phenol Red Sigma P3532-5G
RPMI 1640 ThermoFisher MT10040CM
RPMI 1640 minus glucose VWR 45001-110
Silicon Wafers (100mm) University Wafer 1196
Sodium lactate Sigma L4263-100ML
SU8 2075 Microchem Y111074 0500L1GL
SU8 Developer ThermoFisher NC9901158
Sylgard Elastomer Essex Brownell DC-184-1.1
T75 flasks VWR 82050-856
Triton X-100 Sigma T8787-100ML
TrypLE ThermoFisher 12604021
Trypsin-EDTA (0.5%) ThermoFisher 15400054
Tween20 Sigma P9416-50ML
Y-27632 Stem Cell Technologies 72304
EVG620 Aligner EVG
Plasma cleaner PDC-32G Harrick Plasma
Zeiss AxioObserver Z1 microscope Nikon
Leica SP8 Confocal microscope Leica
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Referencias

  1. Savoji, H., et al. Cardiovascular disease models: A game changing paradigm in drug discovery and screening. Biomaterials. 198, 3-26 (2019).
  2. Patino-Guerrero, A., Veldhuizen, J., Zhu, W., Migrino, R. Q., Nikkhah, M. Three-dimensional scaffold-free microtissues engineered for cardiac repair. Journal of Materials Chemistry B. 8, 7571-7590 (2020).
  3. Breslin, S., O’Driscoll, L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discovery Today. 18, 240-249 (2013).
  4. Pontes Soares, C., et al. 2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression. PLoS One. 7, 38147 (2012).
  5. Jensen, C., Teng, Y. Is it time to start transitioning from 2d to 3d cell culture. Frontiers in Molecular Biosciences. 7, 00033 (2020).
  6. Pinto, A. R., et al. Revisiting cardiac cellular composition. Circulation Research. 118, 400-409 (2016).
  7. LeGrice, I., Pope, A., Smaill, B. . Interstitial Fibrosis in Heart Failure. 253, 3-21 (2005).
  8. Veldhuizen, J., Migrino, R. Q., Nikkhah, M. Three-dimensional microengineered models of human cardiac diseases. Journal of Biological Engineering. 13, 29 (2019).
  9. Agarwal, A., Goss, J. A., Cho, A., McCain, M. L., Parker, K. K. Microfluidic heart on a chip for higher throughput pharmacological studies. Lab Chip. 13, 3599-3608 (2013).
  10. Schaaf, S., et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS One. 6, 26397 (2011).
  11. Zhang, D., et al. Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. Biomaterials. 34, 5813-5820 (2013).
  12. Rao, C., et al. The effect of microgrooved culture substrates on calcium cycling of cardiac myocytes derived from human induced pluripotent stem cells. Biomaterials. 34, 2399-2411 (2013).
  13. Navaei, A., et al. Electrically conductive hydrogel-based micro-topographies for the development of organized cardiac tissues. RSC Advances. 7, 3302-3312 (2017).
  14. Jiang, Y., Park, P., Hong, S. M., Ban, K. Maturation of cardiomyocytes derived from human pluripotent stem cells: Current strategies and limitations. Molecules and Cells. 41, 613-621 (2018).
  15. Yang, X., Pabon, L., Murry, C. E. Engineering adolescence maturation of human pluripotent stem cell-derived cardiomyocytes. Circulation Research. 114, 511-523 (2014).
  16. Kharaziha, M., Memic, A., Akbari, M., Brafman, D. A., Nikkhah, M. Nano-enabled approaches for stem cell-based cardiac tissue engineering. Advanced Healthcare Materials. 5, 1533-1553 (2016).
  17. Ellis, B. W., Acun, A., Can, U. I., Zorlutuna, P. Human iPSC-derived myocardium-on-chip with capillary-like flow for personalized medicine. Biomicrofluidics. 11, 024105 (2017).
  18. Mathur, A., et al. Human iPSC-based cardiac microphysiological system for drug screening applications. Scientific Reports. 5, 8883 (2015).
  19. Mastikhina, O., et al. Human cardiac fibrosis-on-a-chip model recapitulates disease hallmarks and can serve as a platform for drug testing. Biomaterials. 233, 119741 (2020).
  20. Veldhuizen, J., Cutts, J., Brafman, D. A., Migrino, R. Q., Nikkhah, M. Engineering anisotropic human stem cell-derived three-dimensional cardiac tissue on-a-chip. Biomaterials. 256, 120195 (2020).
  21. Truong, D., et al. Human organotypic microfluidic tumor model permits investigation of the interplay between patient-derived fibroblasts and breast cancer cells. Investigación sobre el cáncer. 7, 3139-3151 (2019).
  22. Benam, K. H., et al. Engineered in vitro disease models. Annual Review of Pathology: Mechanisms of Disease. 10, 195-262 (2015).
  23. Karamanova, N., et al. Endothelial immune activation by medin: Potential role in cerebrovascular disease and reversal by monosialoganglioside-containing nanoliposomes. Journal of the American Heart Association. 9, 014810 (2020).
  24. Chen, G., et al. Chemically defined conditions for human iPSC derivation and culture. Nature Methods. 8, 424-429 (2011).
  25. Watanabe, K., et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology. 25, 681-686 (2007).
  26. Narsinh, K. H., et al. Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells. Journal of Clinical Investigation. 121, 1217-1221 (2011).
  27. Cahan, P., Daley, G. Q. Origins and implications of pluripotent stem cell variability and heterogeneity. Nature Reviews Molecular Cell Biology. 14, 357-368 (2013).
  28. Laco, F., et al. Unraveling the inconsistencies of cardiac differentiation efficiency induced by GSKB inhibitor CHIR99021 in human pluripotent stem cells. Stem Cell Reports. 10, (2018).
  29. Rupert, C. E., Kim, T. Y., Choi, B. R., Coulombe, K. L. K. Human cardiac fibroblast number and activation state modulate electromechanical function of hiPSC-cardiomyocytes in engineered myocardium. Stem Cells International. 2020, 9363809 (2020).
  30. Ban, K., Bae, S., Yoon, Y. S. Current strategies and challenges for purification of cardiomyocytes derived from human pluripotent stem cells. Theranostics. 7, 2067-2077 (2017).
  31. Uosaki, H., et al. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression. PLoS One. 6, 23657 (2011).
  32. Dubois, N. C., et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nature Biotechnology. 29, 1011-1082 (2011).
  33. Lian, X., et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nature Protocols. 8, (2013).
  34. Tohyama, S., et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell. 12, 127-137 (2013).
  35. Burridge, P. W., Keller, G., Gold, J. D., Wu, J. C. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell. 10, 16-28 (2012).
  36. Mummery, C. L., et al. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circulation Research. 111, 344-358 (2012).
  37. Radisic, M., et al. Biomimetic approach to cardiac tissue engineering. Philosophical Transactions of the Royal Society of London B Biological Science. 362, 1357-1368 (2007).
  38. Hulsmans, M., et al. Macrophages facilitate electrical conduction in the heart. Cell. 169, 510-522 (2017).
  39. Frantz, S., Nahrendorf, M. Cardiac macrophages and their role in ischaemic heart disease. Cardiovascular Research. 102, (2014).
  40. Truong, D., et al. A three-dimensional (3D) organotypic microfluidic model for glioma stem cells – Vascular interactions. Biomaterials. 198, 63-77 (2019).
  41. Shao, J., et al. Integrated microfluidic chip for endothelial cells culture and analysis exposed to a pulsatile and oscillatory shear stress. Lab Chip. 9, 3118-3125 (2009).
  42. Leclerc, E., Sakai, Y., Fujii, T. Cell culture in 3-dimensional microfluidic structure of PDMS (polydimethylsiloxane). Biomedical Microdevices. 5, 109-114 (2003).
  43. Mukhopadhyay, R. When PDMS isn’t the best. What are its weaknesses, and which other polymers can researchers add to their toolboxes. Analytical Chemistry. 79, 3248-3253 (2007).
  44. van Meer, B. J., et al. Small molecule absorption by PDMS in the context of drug response bioassays. Biochemical and Biophysical Research Communication. 482, 323-328 (2017).
  45. Berthier, E., Young, E. W., Beebe, D. Engineers are from PDMS-land, Biologists are from Polystyrenia. Lab Chip. 12, 1224-1237 (2012).
  46. Chuchuy, J., et al. Integration of electrospun membranes into low-absorption thermoplastic organ-on-chip. ACS Biomaterials Science & Engineering. , (2021).
  47. Soucy, J. R., et al. Reconfigurable microphysiological systems for modeling innervation and multitissue interactions. Advanced Biosystems. 4, 2000133 (2020).
  48. Cutts, J., Nikkhah, M., Brafman, D. A. Biomaterial approaches for stem cell-based myocardial tissue engineering. Biomarker Insights. 10, 77-90 (2015).
  49. Navaei, A., et al. Gold nanorod-incorporated gelatin-based conductive hydrogels for engineering cardiac tissue constructs. Acta Biomaterialia. 41, 133-146 (2016).
  50. Navaei, A., et al. The influence of electrically conductive and non-conductive nanocomposite scaffolds on the maturation and excitability of engineered cardiac tissues. Biomaterial Sciences. 7, 585-595 (2019).
  51. Saini, H., Navaei, A., Van Putten, A., Nikkhah, M. 3D cardiac microtissues encapsulated with the co-culture of cardiomyocytes and cardiac fibroblasts. Advanced Healthcare Materials. 4, 1961-1971 (2015).
  52. Shadrin, I. Y., et al. Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues. Nature Communications. 8, 1825 (2017).
  53. Ronaldson-Bouchard, K., et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature. 556, 239-243 (2018).
  54. Perl, A., Reinhoudt, D. N., Huskens, J. Microcontact Printing: Limitations and Achievements. Advanced Materials. 21, 2257-2268 (2009).
  55. Yu, J., et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 324, 797-801 (2009).
  56. Yu, J., et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 318, 1917-1920 (2007).

Tags

Microfluidic PlatformCardiovascular DiseaseMyocardiumStem Cell-derived CardiomyocytesCardiac FibroblastsCell DissociationCell CultureCell Alignment

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Veldhuizen, J., Nikkhah, M. Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform. J. Vis. Exp. (172), e62539, doi:10.3791/62539 (2021).
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