Name: generation of dynamical environmental conditions using a high-throughput microfluidic device
Uploaded: 2021-04-17T13:00:00
Description: Watch this Scientific Journal Video about Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device at JoVE.com

作者感谢陈氏仪器（中国）有限公司程志峰的技术支持。这项工作得到了资助（中国国家自然科学基金委员会，51927804）。

1. 微流体芯片设计
<ol><li>设计由 18 个入口组成的微流体多路复用器，每个入口都由单个阀门和永久泵控制。为了增加每个泵周期驱动的液体体积，由 3 个控制通道（特意加宽至 200 μm）和 10 条连接的流线组成。</li>
	<li>设计无剪切文化室。二级培养单元的复制由较低细胞培养室（400μm x 400 μm x 150 μm）和较高的缓冲层（400μm x 400 μm x 75 μm）组成，可防止在中等交换期间对细胞产生不必要的剪切?...

<ol>
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各种微流体装置已开发出来，以执行多路复用和复杂的实验17，18，19，20。例如，由一系列拓扑凹槽制成的微井可以捕获单个细胞，而无需使用外部力，表现出有利的特性，包括样本量小、平行、材料成本更低、响应更快、灵敏度高 21、22、23、24。<...

高吞吐量技术对生物医学和临床研究至关重要。通过同时进行数百万次化学、遗传或活细胞和器官测试，研究人员可以快速识别调节生物分子通路的基因，并根据自己的特定需求定制顺序药物输入。机器人1和微流体芯片与设备控制程序相结合，允许复杂的实验程序自动化，包括细胞/组织操作，液体处理，成像和数据处理/控制2，3。因此，根据所需的吞吐量4，5，可以在一个芯片上维持成百上千的实验条件。
在此协议中，我们描述了微流体装置的设计和制造过程，该装置由 1500 个培养单元、一系列增强的围流泵和现场混合模组组成。二级细胞培养室可防止在中等交换过程中不必要的剪切，从而确保长期活细胞成像不受干扰的文化环境。研究表明，拟议中的微流体装置是研究复杂生命机械的高吞吐量的合适平台。此外，微流体芯片的先进功能允许在体内自动重组高度复杂和动态的微环境条件，如不断变化的细胞因子和配体组成6，7，其完成需要几个月的传统平台，如96井板。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2713 Loker Avenue West</td>
			<td>Torrey pines scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AZ-50X</td>
			<td>AZ Electronic Materials, Luxembourg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorotrimethylsilane(TMCS) 92360-25mL</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator HP151</td>
			<td>Heal Force</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Hole Puncher for PDMS chips WH-CF-14</td>
			<td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM(L-glutamine, High Glucose, henol Red)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Balance UTP-313 Max:600g, e:0.1g, d:0.01g</td>
			<td>Shanghai Hochoice Apparatus Manufacturer Co.,LTD.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronection 0.25 mg/mL</td>
			<td>Millipore, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax 100x</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Incubator BGG-9240A</td>
			<td>Shanghai bluepard instruments Co.,Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Model Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep 10 Units/mL Penicillin 10 ug/mL Streptomycin</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner PDC-002</td>
			<td>Harrick Plasma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polydimethylsiloxane(PDMS)</td>
			<td>Momentive</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polylysine 0.01%</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater ARE-310</td>
			<td>Awatori Rentaro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater TDZ5-WS</td>
			<td>Cence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater WH-SC-01</td>
			<td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025</td>
			<td>MicroChem, Westborough, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3075</td>
			<td>MicroChem, Westborough, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of dynamical environmental conditions using a high-throughput microfluidic device

模仿体内环境条件对于复杂生命机械的体外研究至关重要。然而，目前针对活细胞和器官的技术要么非常昂贵，比如机器人技术，要么在液体操作中缺乏纳米线体积和毫秒时间精度。我们介绍一个微型流体系统的设计和制造，该系统由 1，500 个培养单元、一系列增强的永久泵和现场混合模组组成。为了证明微流体装置的能力，神经干细胞（NSC）球体在拟议的系统中保持。我们观察到，当 NSC 球体在第 1 天接触 CXCL，在第 2 天接触 EGF 时，圆形构象维护良好。6 种药物输入顺序的变化导致 NSC 球体的形态变化和 NSC 茎的表达水平代表标记（即 Hes5 和 Dcx）。结果表明，动态复杂的环境条件对NSC分化和自我更新有重大影响，建议的微流体装置是复杂生命机械高吞吐量研究的适宜平台。

传统的芯片上渗透泵最初由斯蒂芬·奎克在2000年描述，使用这种渗透是由模式101，100，110，010，011，0018，10激活的。 数字 0 和 1 表示 3 个水平控制线的"打开"和"关闭"。使用超过3个阀门（例如，5个）的研究也报告了11个。尽管由 3 条控制线和 3 条流线组成的永久泵提供了纳米线精度，但传输速度太慢，无法养活 1，500 个培养室。为了?...

Watch this Scientific Journal Video about Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device at JoVE.com

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

我们介绍了一个用于复杂生命机械高吞吐量研究的微流体系统，该系统由 1500 个培养单元、一系列增强的永久泵和现场混合模组组成。微流体芯片可分析体内高度复杂和动态的微环境条件。

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

微环境使细胞因子、配体浓度和组成具有高度复杂和动态性。创造类似的文化环境对于干细胞分化、免疫反应和芯片平台器官发育等复杂生命机制的体外研究至关重要。但是，使用传统的生物医学方法（如管道处理）很难产生和传递纳米体积和毫秒精度的化学信号。为应对挑战，一个能够替代图像采集生成动态组合和顺序信号输入的文化平台需求量很大。在此协议中，介绍了微流体装置的设计和制造过程。拟议的微流体芯片由 1500 个培养单元、一系列增强的围流泵和现场混合模组组成。我们证明该平台适合研究单细胞二维细胞群和三维神经球体。芯片上复杂动态的环境条件对神经干细胞分化和自我更新有重大影响。设计和制造程序详情如下。芯片设计使用自动CAD软件执行。为了在显微镜上安装板架的尺寸，微型流体芯片的尺寸约为7乘5厘米，由1500个培养室组成。其中一个重要特点是它有由8个流道和200微米宽控制通道组成的渗透泵。与2002年Step stephen Quake提议的永久泵相比，控制与流道之间的重叠面积增加了16倍，达到大约15个纳米升液体和每个泵削周期，足以满足在芯片上平行维持1500个独立环境条件的需要。该设备的另一个重要部分是两层文化室，可以保持无剪切文化环境。通过快速引导它穿过顶层来防止不需要的剪切应力。细胞、组织和关键的有条件介质在底层保持不变。数值模拟表明，当液体通过培养室时，从左到右。剪切力是可以有效预防的。即使在每秒10毫米的输入流速或微米大小的组织保持不受干扰在培养单元的底部。我们使用紫外线光刻技术制造了复制成型或硅晶片。使用 SU-8 3025 负光分辨率产生高度 25 微米和重量 100 微米的流体通道。75微米和150微米高度的培养室是使用SU-8 3075光研究器制造的。AZ50X 正光分辨率用于与控制通道重叠的圆形井功能，以确保良好的连接。为了生产微流芯片，图案和空白硅晶片首先使用TMCS处理。在负0.85兆帕的真空室中，不同数量的单体催化剂比的PDMS 10比1被彻底混合和调试1至2小时。控制层是通过旋转涂层 PDMS 在 2，200 RPM 获得的。硅晶片随后被转移到孵化器，在80摄氏度下孵育60分钟。使用定制光学设备和等离子蚀刻机将不同的层对齐并粘合在一起。入口孔在80摄氏度的热粘合后又打了两个小时。该芯片粘合在 PDMS 涂层盖滑上，与 96 井板大小相同，在使用前在 80 离心层固化至少 12 小时。在操作中，控制通道通过塑料管连接到微型气动电磁阀。所有井的转弯都由自定义 MATLAB 程序通过图形用户界面控制。每个芯片分别确定俯卧撑 PDMS 膜阀的最佳闭合压力，通常范围为 25 到 30 PSI。通过将所有系列井动态组合和顺序输入，可以在芯片上生成。细胞以80%的结合度收获，并以大约每毫升10至6个功率的密度与培养基DMEM一起补充，然后通过加压含有溶液的细胞加载到芯片中。对于神经干细胞的附体和悬浮培养，细胞被收集，球体被直接加载到芯片中。在图像采集方面，使用了带有自动转换阶段的倒置显微镜和数字互补金属氧化物半导体相机。舞台和图像采集通过软件进行控制，显示左上角和右下角腔室作为选择点，并有序地将转换阶段移动到点，以 4 倍目标镜头进行初步成像。将 4 倍目标镜头与 20 倍镜头更改，并调整成像参数，包括光强度、暴露时间等。然后定位选择点，以便每个腔室的位置留下 13 乘 15 个腔室矩阵或由程序自动定义。在确定腔室坐标时，转化阶段移动到每个腔室，其中 X、Y、Z 焦距平面可以微调。设置间隔和成像周期的持续时间。保存路径，然后开始成像。该系统可用于各种目标动态跟踪，如明亮场的神经干细胞分化和神经干细胞球体形成过程中的荧光场。数据分析。使用自定义 MATLAB 程序将第 2 个格式更改为 Tif。并根据点划分数据。要分析的选择对象是细胞或组织。输入阈值、对象大小、噪声大小进行分析。或者您可以选择按步骤分析以优化参数。然后分析所有数据。以 MAT 格式保存结果后，使用绘图绘制结果或痕迹。本文介绍了基于活细胞微环境动态控制光刻技术的高通量微流体芯片系统的规程和文化。我们还描述了一些应仔细控制的参数，例如 PDMS 膜阀的闭合压力和与腔室平行的流体自旋控制。在传统的细胞体外培养中，对细胞培养环境的处理是手工进行的，既费时又费力，充满液体也不容易进行标准控制。特别是几分钟的解决方案。存在产生投入、免疫药物浓度和大量消费等问题。然而，使用我们的微流体系统，可以重复它作为刺激剂在固定剂量的单液等量的均匀浓度，比较不同的培养在不同的文化室在同一时间。不同于一些微型时间分辨率回刺激和子化分辨率通过调节流体。此外，我们的系统可以通过简单的操作获得大量的比较实验结果。我们的系统基于扩散原理，对细胞微环境产生最小的机械干扰。对于更稳定的细胞微环境芯片，您可以增加缓冲层作为培养层深度的列表或减少中等交换的输入流体，即降低压力，这将在这里用于细胞行为的研究。需要注意的是，这项研究只检查了3T3和NSC细胞的培养。对于其他细胞或细胞系，培养条件可与我们的参数相媲美，以调整它。

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

A Microfluidic Device for Studying Multiple Distinct Strains

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

通过高通量微流体压缩系统对多细胞生物进行机械刺激

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...