JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
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Bio-ingénierie

微流体技术，探讨细胞变形

Published: September 03, 2014
doi:
10.3791/51474
, , ,
1Department of Integrative Biology and Physiology,University of California, Los Angeles, 2Department of Aerospace and Mechanical Engineering,University of Notre Dame, 3Molecular Imaging Center,University of Southern California

Summary

我们展示了一个微流体为基础的检测通过微米级的收缩序列来测量时间刻度为细胞转运。

Abstract

在这里，我们详细的设计，制造和使用的微流体设备的评估，以有效地对大量单个细胞的变形能力。通常情况下，可以在1小时实验中获得了10〜2单元中的数据。一种自动图像分析程序使图像数据的高效后实验分析，使处理是在几个小时内完成。我们的设备的几何形状是独特的，因为细胞必须变形，通过一系列的微米尺度收缩，从而使初始变形和各个单元的时间依赖性舒张待测定。此方法对人早幼粒细胞白血病（HL-60）细胞的适用性论证。驱动单元发生变形，通过使用压力驱动的流动微米尺度收缩，我们观察到人类早幼粒细胞（HL-60）细胞中，通过随后的收缩传代更快之前暂时阻塞所述第一收缩为9.3毫秒的平均时间离子，每收缩4.0毫秒的平均中转时间。与此相反，全反式视黄酸处理的（嗜中性粒细胞型）的HL-60细胞通过随后的收缩传代以3.3毫秒的平均传输时间之前，闭塞第一缩颈为只有4.3毫秒。这种方法可以更深入地了解细胞的粘弹性性质，最终揭示这种行为的分子起源。

Introduction

改变细胞形状在许多生物环境是至关重要的。例如，红细胞和白细胞的变形通过毛细血管比自身直径小1。在转移，癌细胞必须变形，通过狭窄的缝隙间，以及曲折的血管和淋巴管网的种子在辅助站点2。探测单个细胞的物理特性，微流控设备提出了可自定义，研究了一系列的细胞行为，包括他们的迁移，通过狭窄的缝隙3，通过微米级的收缩3-9被动变形能力的理想平台。聚二甲基硅氧烷（PDMS）微流体装置是光学透明的，从而使电池变形，以使用光显微镜观察，并使用基本的图像处理工具进行分析。此外，收缩阵列可以被精确定义，同时使多个细胞的分析与吞吐量超过许多现有的技术10,11。

在这里，我们提出了一个详细的实验方案，用于探测使用“细胞变形器”的PDMS微流体装置，细胞变形。该设备被设计成使得通过连续收缩的细胞通路;这种几何结构是在生理环境下，如肺毛细血管床12共同。为了测量细胞变形，运输时间提供了一个容易测量通过一个单一的收缩4,6需要一个单独的单元格过境时的方便度量值。为了保持整个狭窄通道在细胞中转恒定的压力降，我们用压力驱动流。我们的协议包括由压力驱动的流动，制备和细胞的成像，以及图像处理的装置的设计和制造中，设备操作的详细说明，测量时间为细胞变形，通过一系列收缩的。我们有这两个设备的设计和视觉数据处理代码的补充文件。随着数据的代表性样本，我们证明细胞转运时间通过一系列收缩的收缩作为传代次数的函数。时间刻度为细胞转运分析，虽然微流体装置的窄收缩可以揭示在各种细胞类型4,5,13的变形差异。这里展示的设备唯一调查了细胞转运通过一系列的微米级收缩;此设计模拟在循环的细胞经历，也使探测单元的额外的物理特性，如松弛时间的曲折路径。

Protocol

1，微流控装置的设计注：该设备设计有四个基本功能区域：输入端口，细胞过滤器，节流阵列，和出口（ 图1）。整体的设计可应用于细胞类型的广泛，与微调尺寸。这里提供的是连同有效的选择主要和永生化细胞的设备参数的一些基本设计建议。 选择缩颈阵列通道（ 图1B）的宽度为平均泡孔直径的约30-50％;此缩颈至细胞大小比结果显著细?…

Representative Results

研究了不同类型的细胞，人髓性白血病细胞（HL-60），区分中性粒细胞，小鼠淋巴细胞的细胞，和人卵巢癌细胞系（OVCAR8，HEYA8）的变形用“细胞变形器”微流控技术进行评估。对HL-60和中性粒细胞型HL-60细胞的在途时间代表结果表明，该时间表的单细胞中转通过一系列收缩的， 如图6。运输时间测量单个细胞的人口每7微米的收缩在一系列7收缩在28千帕（ 图6）的驱动压力?…

Discussion

下面我们来分析细胞的使用压力驱动流过收缩的微流体通道过境的变形提供了全面的实验过程。用MATLAB脚本实现自动数据处理（补充材料）;该代码的更新版本保持（ www.ibp.ucla.edu/research/rowat ）。更广泛地说，这里提出的技术可以适用于许多基于细胞的微流体分析，包括细胞骨架的24,23的作用和核硬化剂以及测定癌细胞类型4,5的变形。?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者要感谢劳埃德翁建设性投入此技术的早期版本中，杰里米Agresti压力盖设计技巧博士和齐东平博士，他在制造压力盖的帮助。我们感谢M. Teitell和P. Gunaratne的实验室进行测试，提供各种细胞样本。我们感谢美国国家科学基金会（职业奖DBI-1254185），加州大学洛杉矶分校琼森综合癌症中心和加州大学洛杉矶分校临床和转化科学研究所支持这项工作。

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Pluronic F-127 Block Copolymer Surfactant  Fisher Scientific  8409400 Produced by BASF, also available through Sigma
PDMS base and crosslinker Essex Brownell DC-184-1.1 Product commonly named Sylgard 184 Elastomer
Oxygen plasma discharge unit Enercon Dyne-A-Mite 3D Treater
Biopsy Punch, Harris Uni-Core (0.75 mm) Ted Pella, Inc. 15072
Fingertight Ferrule, 1/32" Upchurch Scientific UP-F-113
Fingertight III Fitting, 10-32 Upchurch Scientific UP-F-300X
polyetheretherketone (PEEK) tubing, outer diameter = 1/32"or 0.79 mm Valco TPK.515-25M
polyethylene (PE-20) tubing, 0.043" or 1.09 mm Becton Dickinson 427406
Pressure regulator Airgas or Praxair
Polyurethane tubing, 5/32” OD McMaster Carr 5648K284
Push-to-connect fittings McMaster Carr 5111K91
Voltage to Pressure (E/P) Electropneumatic Converter Omega IP413-020
16-bit,250 kS/S, 80 Analog Inputs Multifunction DAQ National Instruments NI PCI 6225-779295-01
Analog Connector Block-Screw Terminal National Instruments SCB-68-776844-01
LabView System Design Software National Instruments
Matlab Software The MathWorks, Inc. Matlab R2012a Code requires the Image Processing Toolbox
Shielded Cable National Instruments SHC68-68
DOWNLOAD MATERIALS LIST

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Tags

Keywords: Microfluidic DeviceCell DeformabilityCell DeformationPressure-driven FlowImage AnalysisHL-60 CellsAll-trans Retinoic AcidNeutrophil-typeViscoelastic PropertiesMolecular Origins
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Hoelzle, D. J., Varghese, B. A., Chan, C. K., Rowat, A. C. A Microfluidic Technique to Probe Cell Deformability. J. Vis. Exp. (91), e51474, doi:10.3791/51474 (2014).
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