JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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生物工程

发展多深的圆形横截面微内皮化的一个芯片的程序

Published: October 21, 2013
doi:
10.3791/50771
, , ,
1Lane Department of Computer Science and Electrical Engineering,West Virginia University, 2Department of Cell Biology and Neuroscience,University of California at Riverside

Summary

Â微通道上的一个芯片平台开发回流焊的光致抗蚀剂光刻技术，软光刻技术和微流体的结合。模拟的三维（3D）的几何形状在体内微血管内皮化的微通道平台，控制连续灌注流量下运行，允许高品质和实时成像，并可以应用对微血管研究。

Abstract

努力一直专注于发 ​​展在体外实验研究微血管，因为动物体内研究更费时，昂贵，观察和量化是非常具有挑战性的。然而，传统的在体外微血管检测体内微血管代表相对于三维（3D）的几何形状，并提供连续的流体流动的限制。使用回流焊的光致抗蚀剂光刻技术，软光刻技术，以及微流控芯片的组合，我们已经制定了多深的圆形横截面内皮化微的芯片，它模仿3D几何体内微血管和运行控制连续灌注下流程。使用了一个正的回流的光致抗蚀剂，制作母模的半圆形横截面的微通道网络。由对准和粘接的聚二甲基硅氧烷（PDMS）的微通道REPL从主模具icated，的圆柱形微通道网络已创建。微通道的直径可以得到很好的控制。此外，原代人脐静脉内皮细胞（HUVECs）表明，在芯片内接种细胞排列的微通道的内表面上为4天至2周之间的时间周期根据控制灌注持久。

Introduction

微血管，作为流通体系的一部分，调解血液和组织之间的相互作用，支持新陈代谢活动，定义组织微环境，在许多健康和病理条件下发挥了关键作用。再演在体外的功能微血管提供了一个平台，为研究复杂的血管现象。然而，传统的在体外微血管检测，如内皮细胞迁移测定，内皮细胞管腔形成的测定中，大鼠和小鼠的主动脉环测定，无法重新创建的三维（3D）的几何形状和连续的流量控制相对于体内微血管1-8。微血管的研究使用的动物模型中和在体内测定，如角膜血管生成法，鸡胚尿囊膜血管生成实验，Matrigel栓实验，是更费时，成本高，相对于观察和量化挑战，并引发伦理问题1，9-13。

显微制造和微流控芯片技术的进展已启用了各种各样的见解，到生物医学科学在削减高的实验与动物体内研究，如容易和严格控制的生物条件和动态流体环境中，不会有相关的成本和复杂性的同时一直未能与传统的大容量的技术。

在这里，我们提出了一种方法来构建内皮化微通道上的单芯片模拟3D几何体内微血管和控制的连续灌流采用回流焊的光致抗蚀剂光刻技术，软光刻技术和微流体的结合下运行。

Protocol

1。光致抗蚀剂光刻制作模具师傅以下协议显示制程制作的微通道直径在30-60微米之间。为了得到一个更小的直径（小于30微米），一个单一的光致抗蚀剂的旋涂的微通道是必要的。 转移从冰箱中在4℃下回流的光致抗蚀剂的洁净室在使用前24小时，并允许其升温至室温。 清洁硅片上并烘烤1小时，在150℃下使其脱水。脱水将协助光致抗蚀剂的硅衬底的粘附性。 </li…

Representative Results

我们的方法来制造微通道网络的多深度模仿体内微血管的复杂的三维几何形状，其中的微通道具有圆形横截面15。此外，父分支通道的女儿通道的直径近似服从默里定律保持在所需的水平的流体流动，使整体的流路阻力变低，流动速度更均匀的整个网络16-18。 电影1，图2，和电影 ，分别为一个半圆形的光致抗蚀剂制造模具师傅和圆形的横截面PDMS微?…

Discussion

1。主模具制造

血管形态计量学的设计和指导原则之一是被称为穆雷的第16条，其中规定，受整个网络分布的血管直径最小能量代价。报告还指出，在分叉的父容器的直径的立方等于总和的女儿血管直径的立方（ 公式1 19）此外，泊肃叶定律已被用于估?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

部分支持这项研究是由美国国家科学基金会（NSF 1227359），西弗吉尼亚的EPSCoR程序由美国国家科学基金会（EPS-1003907），西弗吉尼亚ADVANCE办公室主办，由美国国家科学基金会（1007978），和WVU PSCoR的分别。 WVU共享研究设施（无尘室设施）和微流控结合蜂窝片上实验室（芯片实验室）在西弗吉尼亚大学的研究工作是在微细。聚焦成像做在WVU显微镜成像设备。

Materials

Reagent/Material
Reflow Photoresist AZ Electronic Materials AZP4620
Developer AZ Electronic Materials AZ 400K
PDMS Dow Corning Corporation Sylgard 184
MCDB 131 Culture Medium Invitrogen 10372-019
NacBlue Nuclei Staining Invitrogen H1399
PKH Red Stain Sigma MINI26 and PKH26GL
Fibronectin Gibco PHE0023
L-Glutamine Sigma G7513
Phosphate Buffered Saline Invitrogen 14040-133
HEPES Buffered Saline Solution Lonza CC-5024
Trypsin/EDTA Invitrogen 25300-062
Trypsin Neutralizing Solution Lonza CC-5002
PDMS Curing Agent Dow Corning Corporation Sylgard 184
Primary Human Umbilical Vein Endothelial Cells Lonza CC-2517
Fetal Bovine Serum Lonza 14-501F
Diluent C Sigma CGLDIL
Hoechst33342 Invitrogen, Molecular Probes R37605
Dextran Sigma 95771
3.5% Paraformaldehyde Electron Microscopy Science 15710-S
Equipment
Spinner Laurell Technologies Corporation WS-400BZ-6NPP/LITE
Desiccator BelArt Products 999320237
Inverted Microscope Nikon Eclipse Ti
Syringe Pump System Harvard Apparatus PHD Ultra
Laminar Biosafety Hood Thermo Scientific 1300 Series A2
Planetary Centrifugal Mixer Thinky ARE-310
Isotemp Oven Fisher Scientific 13-246-516GAQ
Optical Microscope Zeiss Invertoskop 40C
Plasma Cleaner Harrick Plasma PDC-32G
Hotplate Barnstead/Thermolyne Cimarec SP131635
Laser Scanning Confocal Microscope Zeiss LSM 510
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Tags

Keywords: Multi-depth Circular Cross-sectional MicrochannelsEndothelialized MicrochannelsIn Vitro Microvessels3D GeometryContinuous Fluid FlowPhotolithographic ReflowSoft LithographyMicrofluidicsPDMSPrimary Human Umbilical Vein Endothelial Cells (HUVECs)Perfusion
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Li, X., Mearns, S. M., Martins-Green, M., Liu, Y. Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip. J. Vis. Exp. (80), e50771, doi:10.3791/50771 (2013).
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