热 Nanoimprinting 技术制备梯度 Nanopattern 及其对人血管内皮细胞菌落形成的响应筛选
Summary
在这里, 我们提出了一个通过热 nanoimprinting 制备梯度 nanopattern 板的协议, 以及筛选人类内皮祖细胞对纳米结构的反应的方法。通过使用所描述的技术, 有可能产生一个脚手架, 可以操纵细胞行为的物理刺激。
Abstract
Nanotopography 可以在身体周围的各种细胞外基质 (ECMs) 中找到, 并且已知对细胞反应有重要的调控作用。然而, 由于缺乏适当的筛查工具, 很难确定纳米结构的尺寸与细胞的反应之间的关系。在这里, 我们展示了可重现的和经济高效的梯度 nanopattern 板的开发, 用于操作细胞反应。以阳极氧化铝 (氧化铝) 为主要模具, 采用热印迹技术制备了 nanopillars 直径范围增大的梯度 nanopattern 板 [120-200 nm (gp 120/200)、200-280 nm (gp 200/280) 和 280-360 nm (gp 280/360)]。这些梯度 nanopattern 板的设计, 以模仿不同大小的 nanotopography 在 ECM 和被用来筛选的反应, 人类内皮细胞集落形成单元 (hECFCs)。在本协议中, 我们描述了在细胞工程中制作梯度 nanopattern 板的分步过程, 从人外周血中培养 hECFCs 的技术, 以及在 nanopattern 板上培养 hECFCs。
Introduction
近年来, 在细胞工程1、2、3、4等领域, 对表面形貌的物理刺激对细胞的反应关注。因此, 更多的注意力集中在三维纳米结构在细胞附着表面5。据报道, 整合素是细胞的表面识别装置, 通过机械转导6传递了 ECM 微纳米结构驱动的物理刺激。这种机械刺激调节细胞行为通过接触指导7和诱导骨架重组改变形状, 除了病灶粘连和僵硬的细胞8。
人体内皮祖细胞 (hEPCs) 在体内与周围 ECM9的微环境紧密地相互作用。这表明 ECM 的物理状态作为特定细胞基质粘附复合物形成的一个重要参数, 与血流产生的剪应力10有关。据报道, 表面 nanotopography 增强了 hEPCs11毛细管网络的体外形成, ECM/生物可溶性因子联合系统使 hEPCs 能够识别功能失调的基底, 并促进伤口愈合12,13。然而, ECM 和 hEPCs 之间的关系还没有得到明确的理解。
虽然许多研究人员试图澄清细胞反应和物理线索之间的关系, 从不同的基质14,15,16, 这些研究只使用固定尺寸的纳米结构或nanopatterns 与不规则的安排有限制, 以阐明纳米结构和细胞行为的大小之间的关系。这里的问题是缺乏合适的工具来筛选细胞的反应, 可以取代现有的繁琐和迭代的方法, 以找到最佳尺寸的纳米结构。因此, 需要一个简单的技术来筛选细胞反应的物理刺激不重复。
在这里, 我们描述了一个方法, 在我们以前的报告17,18,19产生一个梯度 nanopattern, 其中排列的 nanopillars 直径逐渐增加。此外, 我们还描述了如何培养和分析 hECFCs 在梯度 nanopattern 板上的行为, 以确定物理刺激对细胞的影响。采用轻度阳极氧化、渐进蚀刻和防粘层涂敷的方法, 制备了梯度氧化铝模具。采用热印迹光刻技术, 以成本效益高、简便的方式生产出相同的聚苯乙烯梯度 nanopatterns。利用梯度 nanopatterns, 确定纳米结构的大小对一组实验中的细胞行为有很大的影响是可行的。我们期望这种梯度 nanopattern 有助于理解血液衍生 hECFC 或其他细胞与不同尺寸的纳米结构之间的相互作用机制。
Protocol
这项研究是由韩国大学 Anam 医院的机构审查委员会批准的 (IRB 号。ED170495)。所有程序都是按照《赫尔辛基宣言》及其后来的修正案进行的。 1. 用抛光制备铝 (铝) 基板 警告:抛光溶液具有腐蚀性和毒性。佩戴个人防护用品, 包括丁腈橡胶手套、护目镜和实验室大衣。在通风罩中执行此步骤。 在 60% L 双夹克烧杯 (c1h2oh: HClO…
Representative Results
图 1显示了根据其类型和位置, 制备的梯度氧化铝模具的 SEM 图像。图 2显示了具有正圆 nanopillars 的梯度 nanopattern 板的 SEM 图像,图 3是 nanopillar 直径的量化数据。表 1列出了预制 nanopillars 的特点。 图 4A显示了血液衍生 hECFCs …
Discussion
阳极氧化铝的制备常常受到裂纹、孔隙形状不规则和燃烧等缺陷的影响。这些缺陷的主要原因称为电解击穿, 这是严重影响了金属基板的本质是电化铝和电阻率的电解质21。由于电解质的电阻率随温度的不同而变化, 从电极上连续消除热量是保持电解质在这种高压阳极氧化条件下的位置温度稳定的关键点。在本协议中, 我们通过修改 Masuda 的两步阳极氧化22,</su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了由教育部、科学技术部 (MEST) [NRF-2015R1D1A1A01060397] 资助的韩国国家研究基金会 (NRF) 和生物 & 医疗技术发展的基础科学研究项目的支持。由科学、信息和通信技术 & 未来规划部资助的 NRF 计划 [NRF-2017M3A9C6029563]。
Materials
| Perchloric acid 60% | Daejung Chemicals & Metals | 6512-4100 | |
| Ethyl alcohol, absolute 99.9% | Daejung Chemicals & Metals | 4118-4100 | |
| Phosphoric acid 85% | Daejung Chemicals & Metals | 6532-4400 | |
| Methyl alcohol 99.5% | Daejung Chemicals & Metals | 5558-4400 | |
| Chromium(VI) oxide | Daejung Chemicals & Metals | 2558-4400 | |
| Sulfuric acid 95% | Daejung Chemicals & Metals | 7781-4100 | |
| Hydrogen peroxide 30% | Daejung Chemicals & Metals | 4104-4400 | |
| n-hexane 95% | Daejung Chemicals & Metals | 4081-4400 | |
| Toluene 99.5% | Daejung Chemicals & Metals | 8541-4400 | |
| (heptadecafluoro-1,1,2,2,-tetrahydrodecyl)dimethylchlorosilane | Gelest | SIH5840.4 | Moisture sensitive |
| Methoxynonafluorobutane 99% | Sigma aldrich | 464309 | |
| Collagen solution | Stemcell | #4902 | |
| Gelatin | Sigma aldrich | G1890 | Protein coating solution |
| Ficoll-Paque | GE Heathcare | 17-1440-03 | Hydrophilic polysaccharide solution |
| EGM-2MV | Lonza | CC-3202 | Endothelial cell expansion medium |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| Phosphate buffered saline | Gibco | 10010031 | |
| Fetal bovine serum | Gibco | 12483-020 | |
| Paraformaldehyde | Sigma aldrich | P6148 | |
| Glutaraldehyde | Sigma aldrich | G5882-100ML | |
| Osmium tetroxide | Sigma aldrich | 201030-1G | |
| Hexamethyldisilazane | Sigma aldrich | 440191 | |
| Triton X-100 | Sigma aldrich | X100-100ML | Octylphenol ethoxylate |
| Goat serum | Gibco | 26050-088 | |
| anti-human vinculin primary antibody | Sigma aldrich | V9131 | |
| F-actin probe | Molecular Probes | A12379 | Fluorescence-conjugated phalloidin |
| Alexa Fluor 488-conjugated anti-mouse IgG antibody | Molecular Probes | A11001 | Fluorescence-conjugated secondary antibody |
| 4',6-diamidino-2-phenylindole | Sigma aldrich | D9542 | |
| Mounting medium | DAKO | S3023 | |
| Anti-human vWF primary antibody | DAKO | A0082 | |
| Anti-human CD144 primary antibody | BD Biosciences | #555661 | |
| Eponate 12™ Embedding Kit, with BDMA | Ted Pella | 18012 | Epoxy resin |
| Uranyl Acetate, 25g | Ted Pella | 19481 | |
| Lead Citrate, Trihydrate, 10g | Ted Pella | 19312 | |
| Ultra pure aluminum plate | Goodfellow | 26050-088 | |
| Polystyrene sheet | Goodfellow | ST313120 | |
| 8.0" silicon wafer | Siltron | 29-01024-03 | Single side polished, 725 µm thick |
| Vacuum desiccator, 4.4 L | Kartell | KA.230 | |
| Vacuum pump | Vacuumer | V3.VOP100 | |
| Power supply | Unicorntech | UDP-3003 | |
| Magnetic stirrer | Daihan scientific | SL.SMS03022 | |
| Overhead stirrer | Daihan scientific | HT120DX | |
| Circulator | Daihan scientific | WCR-P12 | |
| Linear moving stage | Zaber | A-LSQ300A-E01-KT07 | |
| Angle bracket, 90 degrees | Zaber | AB90M | Accessory of the linear moving stage |
| PMP forcep, 145 mm | Vitlab | 67995 | Nonmetallic tweezer |
| PTFE beaker, 250 mL | Cowie | CW007.25 | |
| Ultrasonic cleaner | Branson | B2510MTH | |
| PCB cutter | Hozan Tool Industrial | K-110 | |
| Nanoimprint device | Nanonex | NX-2000 | |
| Oxygen plasma generator | Femto Science | CUTE | |
| Low temperature sterilizer | Lowtem | Crystal 50 | |
| CO2 Incubator | Panasonic | MCO-18AC | |
| Confoal laser scanning microscope | Carl Zeiss | LSM700 | |
| Scanning electron microscope | JEOL | JSM6701 | |
| Transmission electron microscope | Hitachi | H-7500 |
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Cite This Article
Kim, D. H., Cui, L., Seo, H., Joo, H. J., Choi, S., Lim, D., Lee, K. B. Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells. J. Vis. Exp. (137), e57661, doi:10.3791/57661 (2018).