使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires
Summary
心脏biowire平台是使用三维细胞培养与电刺激结合成熟人胚胎和诱导的多能干细胞衍生的心肌细胞（HPSC-CM）的体外方法。这份手稿介绍了心脏biowire平台的详细设置。
Abstract
人类多能干细胞衍生的心肌细胞（HPSC-CMS）是一个有前途的细胞来源，并从而鼓励他们在心脏研究潜力的应用，包括药物发现，疾病建模，组织工程和再生医学的研究。然而，由现有的协议产生的细胞显示与天然成年心室肌相比的范围不成熟。许多已作出努力以成熟HPSC-CMS，只有迄今达到中等成熟。因此，工程化的系统，称为biowire，已通过提供物理和电线索导致HPSC-CMS 在体外更加成熟的状态设计的。该系统使用微制造平台种子HPSC-CM的I型胶原沿着一个刚性模板缝合组装成对准的心脏组织（biowire），将其进行电场刺激具有逐渐增加的频率胶凝。相比未刺激控制，刺激biowired心肌细胞表现出的结构和电生理学成熟的增强程度。这样的变化是取决于刺激速率。这份手稿详细描述了biowires的设计和创作。
Introduction
细胞治疗是最有前途和研究战略，以实现心肌修复/再生一个。已经通过心脏组织工程和生物材料1，2的共递送辅助。大多数现有的细胞来源进行了研究，在动物模型为他们的损伤，患病或年老心3潜在的有利影响。尤其显著已经努力用人类多能干细胞（HPSC）衍生的心肌细胞（HPSC-CM），用于心脏组织工程可能无限的自体细胞来源。 HPSC-CMS可以使用多种已建立的方案4，5，6来制造。然而，所获得的细胞相比，成年心室肌7显示胚胎样表型，具有一定范围的未成熟的特性， </sup> 8。这可能是一个障碍HPSC-CMS应用在药物研发和成人心脏疾病模型9的发展成人心脏组织的模型。
为了克服表型不成熟的这种限制，新方法已被积极地研究促进心肌细胞成熟。早期的研究通过循环机械10或电刺激11显露在新生大鼠心肌细胞的有效促成熟性质。凝胶压实和环状机械刺激还显示出改善HPSC-CM熟化12， 图13，与电生理和钙的处理性能的增强最小的某些方面。因此，所谓的“生物丝”（biowire）的平台系统是由同时提供结构线索和电场stimulatio设计n增强hPSC-CMs的成熟14 。该系统使用微制造平台来创建适合于电场刺激的对准的心脏组织。这可以用于改善hPSC-CM的结构和电生理成熟度。在这里，我们描述制作这样的生物线的细节。
Protocol
主设计与制作注意：使用软光刻技术进行器件制造。为二甲基硅氧烷（PDMS）成型制成两层SU-8主机。 使用设计和绘图软件设计设备（ 图1A ，左）。分别绘制主体的每一层。在20,000 dpi的两个光掩模上打印设备设计，对应于主板15的两层。将设备图案设置为透明，周围区域为黑色;掩模将用作通过光刻将器件图案光学转移到SU-8上的模板（如其…
Representative Results
理性在biowires使用的缝合线的是，以作为在一个轴对准并模仿心肌纤维的形状的3D结构的形成的模板。我们发现，之后在biowire培养7天的细胞改造围绕缝合（ 图3A）的凝胶。沿着缝合线的轴线组装细胞形成对准心脏组织（ 图3）。后7天的预培养物，在biowires进行7天电场刺激的，并与心肌细胞的结构和功能的成熟兼容进一步表现出的性质。 <p class="…
Discussion
该手稿描述了改进hPSC-CMs成熟的设计平台的生物线的设置和实现。该装置可以在标准的微细加工设备中制造，并且生物线可以用普通的细胞培养技术和电刺激器生产。
据我们所知，没有报道类似于生物线的方法。该策略表明，改善的成熟性质取决于电刺激方案，如通过较高刺激速率上升方案（6Hz对3Hz）的生物线中获得的较大成熟水平所证明的。生物线中的心肌细胞与培养20天?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由一个赠款的援助从心脏和中风支持加拿大（G-14-0006265）的基础，从健康研究（137352和143066）和普·比克尔基金会授予加拿大学院运营赠款（1013821 ）到SSN。
Materials
| L-Ascorbic acid | Sigma | A-4544 | hPSC-CM culture media componet |
| AutoCAD | Autodesk, Inc | Software to design device | |
| Carbon rods, Ø 3 mm | Electrical stimulator chamber component | ||
| Collagen, type 1, rat tail | BD Biosciences | 354249 | Collagen gel: 2.1 mg/ml of rat tail collagen type I in 24.9 mM glucose, 23.8 mM NaHCO3, 14.3 mM NaOH, 10 mM HEPES, in 1xM199 media with 10 % of growth factor-reduced Matrigel. |
| Collagenase type I | Sigma | C0130 | 0.2% collagenase type I (w/v) and 20% FBS (v/v) in PBS with Ca2+ and Mg2+. Sterilize with 0.22 μm filter and make 12 ml aliquots. Store at -20 °C. |
| Deoxyribonuclease I (DNase I) | EMD Millipore | 260913-25MU | Make 1 mg/ml DNase I stock solution in water. Filter sterile and store 0.5 ml aliquots at −20 °C |
| Drill & drill bits (Ø 1mm and 2 mm) | Dremel | Drill holes in polycarbonate frames | |
| Electrical stimulator | Grass | s88x | |
| Fetal bovine serum (FBS) | WISENT Inc. | 080-450 | |
| D-(+)-Glucose | Sigma | G5767 | Collagen gel component |
| L-Glutamine | Thermo Fisher Scientific | 25030081 | |
| H2O | MilliQ | 18.2 MΩ·cm at 25 °C, ultrapure, to make all solutions | |
| HEPES | Sigma | H4034 | Collagen gel component |
| Hot plate | Torrey Pines | HS40 | |
| Iscove's Modified Dulbecco's Medium(IMDM) | Thermo Fisher Scientific | 12440053 | |
| Mask aligner | EVG | EVG 620 | |
| Matrigel, growth factor reduced | Corning | 354230 | Collagen gel component |
| Medium 199 (M199) | Thermo Fisher Scientific | 11150059 | Collagen gel component |
| Monothioglycerol (MTG) | Sigma | M-6145 | hPSC-CM culture media componet |
| Orbital shaker | VWR | 89032-088 | |
| Penicillin/Streptomycin (P/S) | Thermo Fisher Scientific | 15070063 | |
| Phosphate-buffered saline (PBS) with Ca2+ and Mg2+ | Thermo Fisher Scientific | 14040133 | |
| Plate (6-well) | Corning | 353046 | |
| Plate (6-well), low attachment | Corning | 3471 | |
| Platinum wires, 0.2 mm | Electrical stimulator chamber component | ||
| Polydimethylsiloxane (PDMS) | Dow Corning | Sylgard 184 | |
| Propylene glycol monomethyl ether acetate (PGMEA) | Doe & Ingalls Inc. | To develop the wafer | |
| Pouch, peel-open | Convertors | 92308 | For steam sterilization |
| Silicon wafer, 4-inch | UniversityWafer Inc. | ||
| Sodium bicarbonate (NaHCO3) | Sigma | S5761 | Collagen gel component |
| Sodium hydroxide | Sigma | S8045 | Collagen gel component |
| Sprin coater | Specialty Coating Systems | G3P-8 | |
| StemPro-34 culture medium | Thermo Fisher Scientific | 10639011 | hPSC-CM culture medium. To make 50 ml, add 1.3 ml supplement, 500 μl of 100× L-Glutamine, 250 μl of 30 mg/ml transferrin, 500 μl of 5 mg/ml ascorbic acid, 150 μl of 26 μl /2 ml monothioglycerol (MTG), and 500 μl (1 %) penicillin/streptomycin. |
| Stop media | Wash medium:FBS (1:1) | ||
| SU-8 50 | MicroChem Corp. | photoresist, master component | |
| SU-8 2050 | MicroChem Corp. | photoresist, master component | |
| Transferrin | Roche | 10-652-202 | hPSC-CM culture media componet |
| Trypsin/EDTA, 0.25% | Thermo Fisher Scientific | 25200056 | hPSC-CM culture media componet |
| Wash medium | IMDM containing 1% Penicillin/Streptomycin |
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Cite This Article
Sun, X., Nunes, S. S. Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation. J. Vis. Exp. (123), e55373, doi:10.3791/55373 (2017).