的剪切粘合地图使用SynVivo合成微血管网络代
Summary
Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.
Abstract
细胞/颗粒附着测定是了解参与了疾病的病理生理生化相互作用的关键，并有在追求新颖的治疗学的发展的重要应用。使用静态条件下测定无法捕捉粘附于剪切的依赖性，限制了其在体内环境的相关性。该量化生理流体流动下黏附平行平板流动腔室需要多个实验进行的剪切粘合地图的产生。另外，它们并不代表本体内规模和形态以及需要的试剂的大体积（〜毫升），用于实验。在这项研究中，我们证明了剪切粘合地图的生成使用基于微流体装置，SynVivo-SMN微血管网络中的一个实验。这个装置重现体内脉管系统的复杂的包括几何尺度，形态元素，流动特性和在细胞相互作用体外格式，从而提供基本的生物学现实环境中，在细胞行为，药物输送和药物发现应用研究。证实该检测方法通过研究2μm的生物素包被的颗粒与微芯片的抗生物素蛋白包被的表面的相互作用。在一个单一的检测使粘着与剪切地图为在生理条件下的颗粒，得到的剪切在微血管观察到的整个范围。
Introduction
电流测定来研究细胞 – 细胞和粒子与细胞的相互作用典型地涉及在其中的颗粒或细胞被培养于基质蛋白或细胞贴壁静态孔板形式。在指定的温育时间结束时，粘附的颗粒或细胞的数量正在使用显微镜1量化。尽管这些测定提供显著洞察这些相互作用背后的生化过程，一个关键限制是缺乏生理流体流（典型的微循环）和其上的粒子粘附的影响。
为了克服这一限制， 在体外流动室已被开发，近年来。这些流室常见的元素是灌注在低雷诺数相匹配的血管观察体内 2壁面剪切率的透明设备。血管壁的上的流量C一个表面模拟了生物分子的任涂层或细胞生长汉伯3。颗粒4-7或8-16细胞中，然后流入在流率的所需范围来量化在各种剪切速率下附着粒子的数量。
然而，使用平行板流动室来研究和验证生物化学现象是相当昂贵和费时的。这主要是由于这样的事实：多个实验需要被用于产生所述流体剪切力与附着粒子/细胞数目的地图进行。此外，平板流室需要大量的试剂由于其大尺寸（高度> 250微米，宽度> 1毫米）。最后，这些设备没有精确地模拟几何特征（ 例如 ，分叉）和流量条件（ 如 ，会聚与发散流）存在于体内 。
基于光刻微细17-19的最新进展，加快实验室上的一个芯片领域设备20-21。这些设备已经在开发工具的平行板流动腔与在微米政权尺寸的小型化版本。在尺寸减少量也在试剂，细胞或所需的实验粒子的体积方面产生显著效益。然而，目前可用的设备的一个关键限制是使用线性信道模型微血管，其中不模仿复杂的微脉管系统的体内观察到的。
最近，我们开发了一种新颖的方法，用于重建微血管网络上的一次性塑料基板造成的体内条件下合成的表示。这些设备被称为SynVivo – 合成微血管网（SMN）采用基于软光刻工艺的PDMS发展。 SynVivo-SMN设备可用于获得细胞/颗粒附着22剪切粘合地图，研究靶向给药23和hAVE被证实在体内对数据24-25。在本文中，我们提出了一个协议，使一代剪切粘合地图从体积小到1-5微升从而导致显著节省资源和时间的一次实验。
Protocol
Representative Results
Discussion
平行平板流动腔室，同时提供显著见解细胞 – 细胞和细胞 – 颗粒相互作用，存在一些限制，如高消耗的试剂和需要多次实验中，以产生剪切粘合地图。使用SynVivo -合成微血管网络（SynVivo -脊髓运动神经元）的使之从模仿条件在体内条件下一次实验的剪切附着力地图的生成。此外，显著节省（> 95％）的试剂也得到。
在运行的粒子黏附实验SynVivo-SMN最重要的一步是确保?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
SynVivo技术被授予下＃2R44HL076034从NHLBI发展。
Materials
| SynVivo-SMN | CFD Research | SMN-001 | Exclusive at CFDRC |
| CFD-ACE+ | ESI Inc. | N/A | |
| Avidin | Invitrogen | 43-4401 | Any avidin source will work for this assay |
| Biotinylated Particles | Polysciences | 24173-1 | Any source of biotinylated particles will work for the assay |
| Tygon Tubing | VWR | 63018-044 | Size is typical for use with SynVivo-SMN |
| NIKON Elements | NIKON Instruments | N/A | Any other imaging software can be used |
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