发展与表征<em>体外</em>微血管网络和内皮的定量测量的[Ca<sup> 2+</sup>]<sub>我</sub>和一氧化氮含量
Summary
Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.
Abstract
内皮细胞（EC）衬血管壁体内经常暴露于流动，但培养的内皮细胞通常在静态条件下生长并表现出促炎性表型。虽然微流体装置的发展已被工程师超过二十年被接受，其生物学应用仍然有限。通过生物 应用验证更生理有关体外模型微血管重要的是推动该领域和桥体之间在体外研究的空白。这里，我们提出的详细程序为培养的微血管网络的使用微流体设备与长期灌注能力的发展。我们还使用共聚焦和常规荧光显微镜表明实时其在EC的[Ca 2+] i和一氧化氮（NO）产生的激动剂诱导的变化的定量测量的应用。所形成的微血管网连续灌注工作表明内皮之间发达结。 VE-钙粘蛋白分布更接近在完整微血管比静置培养乳油单层观察。 ATP诱导的EC瞬时增加钙离子浓度 i和NO的产生进行了定量的单个细胞的水平，这验证了培养微血管的功能进行测量。此微流体装置允许内皮细胞到良好控制，生理学相关的流，这使得细胞培养环境比更接近于体内在以往的，静态的2D培养下生长。所述微通道网络的设计是高度通用的，并且制造工艺简单，重复性好。该装置可以方便地集成到共焦的或常规的显微系统，使高分辨率成像。最重要的是，因为培养的微血管网络可以通过初级人内皮细胞来形成，这种方法将作为一个有用的工具来调查如何从病理患者样本改变血液成分影响人类内皮细胞，并提供深入了解临床的问题。它还可以被开发作为用于药物筛选的平台。
Introduction
内皮细胞（EC）衬血管壁体内经常暴露于流动，但培养的内皮细胞通常在静态条件下生长并表现出促炎性表型1,2。微流体技术使得精确地控制流体通过几何约束微尺度（亚毫米）通道3，其提供了用于培养细胞的机会，特别是对血管内皮细胞，所需的流量的条件下生长。这些特点使得比常规的，静态的2D细胞培养更接近体内细胞培养条件。它们是当微流体装置用于模拟不同类型的脉管的，并研究EC响应机械和/或化学的刺激非常重要的。
尽管微通道网络在一个静态的细胞培养，生物医学F中的适应和微流体的应用表现出的优势ield仍然是有限的。通过最近的一篇综述报道，大部分该领域（85％）的出版物仍然是工程类期刊4。微流体装置的性能尚未足够的说服力大多数生物学家从当前技术切换诸如Transwell小测定和宏观尺度培养皿/载玻片该小型化的设备。微流控是一个多学科领域，需要跨学科的合作，以推动这一领域。本技术本文的目的是减少知识缺口学科之间，使制造程序可理解的生物学家，同时提供生物应用和微流体微血管功能验证。可视化实验方案包括微流体装置及其生物实用工具，它代表的工程师和生物学家之间的密切合作制造。
我们最近的一些报道使用体外微血管网络与微流体装置5生物学应用。为了适当地设计所述微通道网络的尺寸和应用所需的剪切应力，数值模型用计算流体动态软件密切估算流动轮廓建造。原代人脐静脉内皮细胞（HUVECs），该接种到达到汇合的微通道， 即覆盖所述微通道的整个内表面上，在3-4天连续灌注。适当的阻挡层形成通过VE-钙粘蛋白染色显示，并与静态细胞培养条件下，并在完整的微血管形成的比较。通过施加在单独灌注完好微血管6-8开发的实验方案，我们定量测量在EC的变化的[Ca 2+] i和一氧化氮（NO）产生的荧光响应于三磷酸腺苷（ATP）的修复作用和共聚焦和常规荧光显微镜。在欧共体激动剂诱导增加[ 钙 离子浓度和NO据报道，作为在微血管通透性6-15炎性介质引起的必要增加细胞内的信号。虽然以前的一些研究表明DAF-2 DA加载微流体装置16,17的图像，合适的分辨率和数据分析还没有达到18。据我们所知，这项研究表明内皮[ 钙 离子浓度和NO利用微流体为基础的系统激动剂诱导动态变化的第一个定量测量。
微细加工技术，灵活地制造微下降到几微米和使复杂的图案的发展，以模仿体内微血管的几何形状。在这里，我们提出了一个典型的微通道网络，三级分支。这个网络是由在一个微细洁净室和软光刻进行光刻的组合制成。
Protocol
1.微流体设备制造一个SU-8 50母模标准光刻制造清洁前旋涂在硅晶片。冲洗，用丙酮进行15分钟，然后用异丙醇（IPA）15分钟的裸2英寸的硅晶片。通过在150℃下将其放置在热板上进行1小时脱水晶片。脱水后，冷却，在室温下的晶片。 旋涂用的SU-8光致抗蚀剂的硅晶片。加2ml的SU-8光致抗蚀剂到晶片上。斜坡晶片在100转/秒的加速度，持续10秒以500rpm，接着在300转/秒的加速度以1000rpm?…
Representative Results
本节展示了一些与此协议开发的培养微血管网络所取得的成果。所述微通道图案是一个三级分支网络（ 图1A）。在此设计中，一个159微米宽的母信道分支成两个126微米宽的通道，和分支再次分成四个100微米宽女儿频道。进行三维数值模拟来估计的0.35微升/分钟的流速（ 图1B），这表明这微通道网络内的三个不同级别的剪切应力下的剪切应力分布。?…
Discussion
在这篇文章中，我们提出详细的协议为培养微血管网络的发展，EC路口和细胞骨架F-肌动蛋白分布特征，以及EC的定量测量钙离子浓度 且使用微流体装置NO的产生。灌注的微流体装置提供的体外模型，其允许在体内微血管几何形状和剪切流动状况的密切模拟。由于培养的微血管网络可以通过初级人内皮细胞来形成，这种方法可以作为一种有用的工具，调查从患者样…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国家心脏，肺和血液研究所授予HL56237，糖尿病和消化研究所和肾脏疾病研究所DK97391-03，美国国家科学基金会（NSF-1227359和EPS-1003907）。
Materials
| ATP | Sigma-Aldrich | A2383 | |
| Acetone | Fisher Scientific | A929 | |
| Biopsy punch | Miltex | 33-31 AA | |
| Bovine Albumin | MP Biomedicals | 810014 | |
| Bovine Brain Extract (BBE) | Lonza | CC-4098 | |
| Cover-slip | Fisher Scientific | 12-542C | |
| DAF-2 DA | Calbiochem | 251505 | |
| Dextran | Sigma-Aldrich | 31390 | |
| Donkey anti-Goat IgG (H+L) Secondary Antibody | Life technologies | A-11055 | |
| DPBS, no calcium, no magnesium | Gibco | 14190-250 | |
| DRAQ5 (nuclei staining) | Cell Signaling Technology | 4084 | |
| Endothelial Cell Growth Supplement (ECGS) | Sigma-Aldrich | E2759-15MG | |
| Fetal Bovine Serum | Gibco | 16000-044 | |
| Fibronectin | Gibco | PHE0023 | |
| Fluo-4 AM | Life technologies | F-14201 | |
| Gelatin from porcine skin | Sigma-Aldrich | G1890-100G | |
| Gentamicin (50 mg/mL) | Gibco | 15750-078 | |
| Glass coverslip | Fisher Scientific | 12-548B | |
| Glass Pasteur pippette | VWR | 14672 | |
| Heparin sodium salt from porcine intestinal mucosa | Sigma-Aldrich | H3393-10KU | |
| HEPES Buffered Saline Solution | Lonza | CC-5024 | |
| Human umbilical vein endothelial cells (HUVECs) | Lonza | CC-2517 | |
| Isopropyl alcohol (IPA) | VWR | 89125 | |
| L-Glutamine (200 mM) | Gibco | 25030-081 | |
| Mammalian Ringer Solution Ingredient | |||
| NaCl (132 mM) | Fisher Scientific | S671-3 | |
| KCL (4.6 mM) | Fisher Scientific | P217-500 | |
| CaCl2 · 2H2O (2.0 mM) | Fisher Scientific | C79-500 | |
| MgSO4 ·7H2O (1.2 mM) | Fisher Scientific | M63-500 | |
| Glucose(5.5 mM) | Fisher Scientific | BP350-1 | |
| NaHCO3 (5.0 mM) | Fisher Scientific | S233-500 | |
| Hepes Salt (9.1 mM) | Research Organics | 6007H | |
| Hepes Acid (10.9 mM) | Research Organics | 6003H | |
| MCDB 131 Culture Medium | Life technologies | 10372-019 | |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| Phalloidin (F-actin staining) | Sigma-Aldrich | P1951 | |
| Phosphate Buffered Saline | Life technologies | 14040-133 | |
| Polydimethylsiloxane (PDMS) | Dow Corning Corporation | Sylgard 184 | |
| Scalpel | Exel Int | 29552 | |
| Scotch tape | 3M | 34-8711-3070-3 | |
| Silicon wafer | VWR | 14672 | |
| SU-8 photoresist | MicroChem | SU-8 2050 Y111072 | |
| SU-8 developer | MicroChem | Y020100 | |
| tissue culture flasks | Sigma-Aldrich | Z707503-100EA | |
| Triton X-100 | Chemical Book | T6878 | |
| Trypsin Neutralizer solution | Gibco | R-002-100 | |
| Trypsin/EDTA Solution (TE) | Gibco | R-001-100 | |
| Tubing | Cole-Parmer | PTFE microbore tubing, 0.012" ID x 0.030" OD | |
| VE-cadherin | Santa Cruz Biotechnology | SC-6458 | |
| Name of Equipment | Company | Catalog Number | Comments/Description |
| Biosafety Laminar hood | NuAire | NU-425 Class II, Type A2 | |
| CCD camera | Hamamatsu | ORCA | |
| Confocal microscope | Leica | TCS SL | |
| Desiccator | Bel-Art | F42022 | |
| Hotplate | Wenesco | HP-1212 | |
| Incubator | Forma Scientific | 3110 | |
| Isotemp oven | Barnstead | 3608-5 | |
| Lithography bench | Karl Suss | MA6 Contact Lithography | |
| Optical microscope | Nikon | L200 ND & Diaphod 300 | |
| Shutter for the CCD camera | Sutter Instrument | Lambda 10-2 | |
| Plasma cleaner | PVA TePla/Harrick plasma | M4L/PDC-32G | |
| Spin coater | Brewer Science | Cee 200X | |
| Syringe pump system | Harvard Apparatus | 703005 | |
| Name of Software | Company | Catalog Number | Comments/Description |
| CAD software | Autodesk | AutoCAD 2015 | |
| CFD simulation software | COMSOL | COMSOL Multiphysics 4.0.0.982 | |
| Images acquire and analyse for NO production | Universal Imaging | Metafluor |
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Citar este artigo
Xu, S., Li, X., Liu, Y., He, P. Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production. J. Vis. Exp. (111), e54014, doi:10.3791/54014 (2016).