模仿神经血管化初始事件的微流体模型
1Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Chinese Education Ministry, School of Biological Science and Medical Engineering,Beihang University, 2School of Engineering Medicine,Beihang University, 3Artificial Intelligence Key Laboratory of Sichuan Province, School of Automation and Information Engineering,Sichuan University of Science and Engineering, 4Beijing Research Center of Urban System Engineering
Summary
在这里，我们提供了一个微流体芯片和一个自动控制的、高效的循环微流体系统，它回顾了血管化的初始微环境，使内皮细胞（ECs）能够同时受到高光泽剪切应力、跨内皮流动的生理水平和各种血管内皮生长因子（VEGF）分布的刺激。
Abstract
神经血管化通常从现有的正常血管中初始化，初始阶段内皮细胞（ECs）的生物力学微环境与下一个神经血管化过程有显著差异。虽然有很多模型来模拟神经血管化的不同阶段，但仍然缺乏在正常血管微环境的相应刺激下投降神经血管化初始过程的 体外 3D模型。在这里，我们重建了一个 体外 3D模型，模仿神经血管化（MIEN）的初始事件。MIEN 模型包含微流体发芽芯片和自动控制、高效循环系统。形成了一个功能齐全、可喷水的微通道，涂有内皮，并在微流体发芽芯片中模拟发芽过程。神经血管化的最初生理微环境通过微流体控制系统进行回顾，通过该系统，IC将同时暴露在高光泽剪切应力、生理性转皮流和各种血管内皮生长因子（VEGF）分布中。MIEN模型可以很容易地应用于神经化机制的研究，并作为药物筛选和毒理学应用的低成本平台具有潜在的前景。
Introduction
神经血管化发生在许多正常和病理过程1，2，3，4，其中包括成人的两个主要过程，血管生成和动脉生成5。除了最知名的生长因子，如血管内皮生长因子（VEGF）6，机械刺激，特别是血流诱发的剪切应力，在调节血管化7方面非常重要。如我们所知，切变应力的大小和形式在血管的不同部位变化很大，动态变化，对血管细胞8、9、10、11、12有重要影响。先前的研究表明，剪切应力可能影响ECs的各个方面，包括细胞表型变化、信号转导、基因表达，以及与壁画细胞13、14、15、16、17、18、19、20的交流：因此，调节神经血管化21，22，23，24。
因此，为了更好地了解神经血管化，在体外重建自然细胞微环境的过程非常重要。最近，利用微制造和微流体技术的进步，建立了许多模型，以制造微型容器，并精确控制微环境25、26、27。在这些模型中，微容器可以产生水凝胶28，29，多二甲基硅氧烷（PDMS）微流体芯片30，31，32或3D生物打印33，34。微环境的某些方面， 如光切变应力22、23、35、36、转肠皮流37、38、39、40、生化梯度血管因子41、42、应变/拉伸43、44、45，并与其他类型的细胞共同培养32、46已被模仿和控制。通常，使用大型储水器或注射器泵提供灌注介质。这些模型中的跨内皮流是由水库和微管22、23、38、40之间的压力下降造成的。然而，机械微环境很难以这种方式持续保持。如果使用具有高剪切应力的高流速进行注水，横皮流将增加，然后超过生理水平。先前的研究表明，在血管化初期，由于完整的ECs和地下室膜，转皮流的速度非常低，通常低于0.05μm/s8。同时，虽然血管系统中的光刻剪压力差异很大，但平均值为5-20 dyn/cm2、11、47，相对较高。目前，以往作品的转肠皮流速一般保持在0.5-15微米/s 22、38、39、40之间，光切变应力一般在10~223以下。不断使ECs同时暴露在高光泽剪切应力和跨内皮流动的生理水平中仍然是一个困难的课题。
在本研究中，我们描述了一个 体外 3D模型，以模拟神经血管化（MIEN）的初始事件。我们开发了微流体芯片和自动控制、高效循环系统，形成注水微管，模拟发芽过程48。通过 MIEN 模型，首先对神经血管化初期刺激的 IC 微环境进行了重新概括。EC 可以同时受到高光泽剪切应力、跨内皮流的生理水平和各种 VEGF 分布的刺激。我们详细描述了建立MIEN模型的步骤和需要注意的要点，希望为其他研究人员提供参考。
Protocol
1. 晶圆准备 注：此协议是针对本研究期间使用的 SU-2075 负光抗光片的特异性。 用甲醇和异丙醇在旋转涂层上清洁硅晶圆 3 到 5 次如下：先在 500 rpm 下旋转 15 s，然后在 3，000 rpm 下旋转 60 s。 将硅晶圆转移到热板上，热板预热至 180 °C，并将晶圆烘烤 10 分钟。 将硅晶圆从热板中取出，冷却至室温。在继续旋转涂层之前，用压缩空气再次清洁晶圆。将 SU-8 …
Representative Results
体外3D模型，以模拟神经血管化（MIEN）的初始事件在这里提出包括一个微流体发芽芯片和一个微流体控制系统。微流体发芽芯片是从以前的出版物22，23，37，40，51，52，53优化。简言之，它包含三个通道和六个端口?…
Discussion
长期以来，对神经血管化的实时观测一直是个问题。最近已开发出几种方法，以制造与ECs衬砌的香水容器，并毗邻细胞外矩阵，以发芽22，32，40，46，54，但机械微环境仍然难以持续。模仿ECs的初始生物力学微环境仍然是一个困难的课题，这些环境受到高光泽剪切应力和低速?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学研究基金会的资助（赠款号11827803，31971244，31570947，11772036， 61533016、U20A20390、32071311）、中国国家重点研究开发计划（2016年YFC1101101和2016YFC1102202）、111项目（B13003）和北京自然科学基金（4194079）。
Materials
| 0.25% Trypsin-EDTA | Genview | GP3108 | |
| Collagen I, rat tail | Corning | 354236 | |
| DAPI | Sigma-Aldrich | D9542 | |
| Electromagnetic pinch valve | Wokun Technology | WK02-308-1/3 | |
| Endothelial cell medium (ECM) | Sciencell | 1001 | |
| Fetal bovine serum (FBS) | Every Green | NA | |
| Fibronectin | Corning | 354008 | |
| FITC-dextran | Miragen | 60842-46-8 | |
| Graphical programming environment | Lab VIEW | NA | |
| Image editing software | PhotoShop | NA | |
| Image processing program | ImageJ | NA | |
| Isopropanol | Sigma-Aldrich | 91237 | |
| Lithography equipment | Institute of optics and electronics, Chinese academy of sciences | URE-2000/35 | |
| Methanol | Sigma-Aldrich | 82762 | |
| Micro-peristaltic pump | Lead Fluid | BT101L | |
| Micro-syringe pump | Lead Fluid | TYD01 | |
| Oxygen plasma | MING HENG | PDC-MG | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| PBS (10x) | Beyotime | ST448 | |
| Permanent epoxy negative photoresist | Microchem | SU-8 2075 | |
| Phenol Red sodium salt | Sigma-Aldrich | P5530 | |
| Polydimethylsiloxane (PDMS) | Dow Corning | Sylgard 184 | |
| Poly-D-lysine hydrobromide (PDL) | Sigma-Aldrich | P7886 | |
| Polytetrafluoroethylene | Teflon | NA | |
| Program software | MATLAB | NA | |
| Recombinant Human VEGF-165 | StemImmune LLC | HVG-VF5 | |
| Sodium hydroxide (NaOH) | Sigma-Aldrich | 1.06498 | |
| Stage top incubator | Tokai Hit | NA | |
| SU-8 developer | Microchem | NA | |
| Trichloro(1H,1H,2H,2H-perfluorooctyl)silane | Sigma-Aldrich | 448931 | |
| TRITC Phalloidin | Sigma-Aldrich | P5285 |
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