Name: a microfluidic platform for stimulating chondrocytes with dynamic compression
Uploaded: 2019-09-13T13:00:00
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression at JoVE.com

我们感谢克里斯托弗·莫雷斯和斯蒂芬·莫林博士对设备设计和制造的支持。这项研究得到了内布拉斯加大学林肯分校（UNL）和内布拉斯加大学医学中心（UNMC）的人类健康生物工程资助，并得到了NIH/NIAMS的AR070242资助。我们感谢内布拉斯加大学医学中心高级显微镜核心设施的Janice A. Taylor和James R. Talaska为共聚焦显微镜提供帮助。

注：在本协议的每一步都佩戴个人防护设备（PPE），如手套和实验室外套。
1. 主模具制造
注：在烟机罩中执行步骤 1.1 - 1.3。
<ol><li>玻璃处理 注：为步骤 1.1 佩戴面罩、手套和实验室外套。<ol>
<li>通过混合硫酸（H2SO4）和过氧化氢（H2O2），使皮兰哈溶液（60 mL）的体积比为3：1。 注意：由于爆炸危险，请勿在同一烟?...

<ol>
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D., Stokes, I. A. 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为了测试压缩应力对生长板软骨细胞的影响，我们开发了微流体软骨细胞压缩装置（图1），用于将不同级别的压缩应力应用于3D藻酸水凝胶支架中的软骨细胞高吞吐量方式的文化。为了帮助其他研究人员采用我们的设备或开发类似的设备，我们在本协议文章中提供了设备制造步骤的详细信息。
该协议的关键步骤是 1） 使用气动微通道（第 1 层）制造 PDMS...

微工程平台是研究分子、细胞和组织水平生物学的宝贵工具，因为它们能够动态控制物理和化学微环境1、2、3 ，4，5，6，7，8.因此，可以同时以严格控制的方式测试多个假设。在生长板软骨的情况下，有越来越多的证据表明，通过作用调节骨骼生长，压缩应力在生长板软骨9、10、11、 12，13，14，15，16，17，18，19，20 21，22，23，24，25。然而，对压缩应力的作用机制——特别是应力如何指导生长板中软骨细胞柱的形成——知之甚少。
该协议的目标是创建一个气动微流体软骨细胞压缩装置26，以阐明生长板软骨细胞中的机械生物学机制（图1a-c）。该装置由两部分组成：气动驱动装置和藻酸盐凝胶结构。微流体气动驱动装置是利用聚二甲基硅氧烷（PDMS）在光和软光刻的基础上制造的。本装置包含 5 x 5 阵列的薄 PDMS 膜气球，可根据其直径不同地膨胀。藻酸盐凝胶结构由嵌入在5 x 5阵列的藻酸盐凝胶圆柱中的软骨细胞组成，整个藻酸-软骨细胞结构与驱动单元一起组装。藻酸盐凝胶结构由气动膨胀的PDMS气球压缩（图1b）。微流体装置根据PDMS气球直径的差异，在单个平台上可同时产生五种不同级别的压缩应力。因此，在多种压缩条件下进行软骨细胞中粒体生物学的高通量试验是可能的。
该协议中描述的微流体器件比传统压缩装置（如外部固定器14、21、23和宏观压缩装置16）具有许多优点。19,27,28用于研究软骨细胞微粒生物学：1） 微流体装置具有成本效益，因为它消耗的样品体积比宏观压缩装置小;2） 微流体装置是有效的，因为它可以测试多个同时压缩条件，3） 微流体装置可以通过在微通道中有限混合的基础上形成化学品的浓度梯度来结合机械和化学刺激，4） 各种显微镜技术（延时显微镜和荧光共聚焦显微镜）可与透明PDMS制成的微流体器件一起应用。
我们采用并修改了Moraes等人7、29的方法，在单个装置中产生不同的压缩应力水平，从而对软骨细胞压缩进行高通量的中度生物学研究。我们的方法适用于需要三维（3D）培养环境的细胞（如软骨细胞）和压缩细胞后的生物测定。虽然一些微流体细胞压缩装置可以压缩在二维（2D）基板上培养的细胞30，31，32，但它们不能用于软骨细胞，因为2D培养的软骨细胞去区分。有微流体平台用于压缩光聚合水凝胶7、33中的3D培养细胞，但由于从光聚合中分离细胞，压缩实验后分离细胞的微流体平台有限水凝胶是不容易的。此外，可能需要评估紫外线 （UV） 暴露和照片交联启动器对细胞的影响。相反，我们的方法允许在压缩实验后快速分离细胞进行后生物测定，因为藻酸盐水凝胶可以通过钙包剂快速去聚。详细的器件制造和表征方法在本协议中进行了描述。图2显示了制造微流体软骨细胞压缩装置的简要程序。

<table>
	<tbody>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane (ATPES)</td>
			<td>Sigma-Aldrich</td>
			<td>741442-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>(Tridecafluoro-1, 1, 2, 2-Tetrahydrooctyl)-1-Trichlorosilane</td>
			<td>United Chemical Technologies</td>
			<td>T2492-KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K354</td>
			<td></td>
		</tr>
		<tr>
			<td>Air pump</td>
			<td>Schwarzer Precision</td>
			<td>SP 500 EC-LC4.5V DC</td>
			<td>We used the model purchased in 2015. The internal design and performance of air pump (SP 500 EC-LC) changed in early 2016. Also, air pump performance has changed in the course of time. Thus, air pressure generated by an SP 500 EC-LC air pump should be calibrated before use.</td>
		</tr>
		<tr>
			<td>Alginate powder</td>
			<td>FMC Corporation</td>
			<td>Pronova UP MVG</td>
			<td></td>
		</tr>
		<tr>
			<td>Barb Straight Connectors (Metal tube)</td>
			<td>Pneumadyne</td>
			<td>EB40-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Invitrogen</td>
			<td>C3100MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dyed red aqueous fluorescent particles</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>22980</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam pad</td>
			<td>GRAINGER</td>
			<td>Item # 5GCE8</td>
			<td></td>
		</tr>
		<tr>
			<td>Function / Arbitrary Waveform Generator</td>
			<td>Keysight Technologies</td>
			<td>33210A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Chemical</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Fisher BioReagents</td>
			<td>BP2633500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>BDH1174-4LP</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-267-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Keysight Technologies</td>
			<td>E3630A</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Chemical</td>
			<td>S318-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Solenoid manifold</td>
			<td>Pneumadyne</td>
			<td>MSV10-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Solenoid valve</td>
			<td>Pneumadyne</td>
			<td>S10MM-30-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies</td>
			<td>WS-650Mz-23NPPB</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8 Developer</td>
			<td>MicroChem Corp.</td>
			<td>Y020100 4000L1PE</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-100</td>
			<td>MicroChem Corp.</td>
			<td>Y131273 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-5</td>
			<td>MicroChem Corp.</td>
			<td>Y131252 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>Thermo Fisher Scientific</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>EMD Millipore</td>
			<td>MSX12445</td>
			<td></td>
		</tr>
	</tbody>
</table>

a microfluidic platform for stimulating chondrocytes with dynamic compression

众所周知，机械刺激可以调节细胞和组织的生物功能。最近的研究表明，压缩应力会改变生长板软骨结构，导致儿童长骨的生长调节。为了确定压缩应力在骨骼生长中的作用，我们创建了一种由气动压力驱动微流体装置，以动态（或静态）压缩嵌入在藻酸盐水凝胶缸中的生长板软骨细胞。在本文中，我们将介绍制造和描述此设备的详细方法。我们的协议的优点是：1） 在单个平台上的五个技术复制上可以生成五种不同幅度的压缩应力;2） 通过传统的光学显微镜很容易可视化细胞形态;3） 细胞可以快速分离从设备压缩后，以方便下游检测，4）该平台可应用于研究任何细胞类型的机械生物学，可以在水凝胶中生长。

本文介绍了微流体软骨细胞压缩装置制造的详细步骤（图2）。该器件包含5×5阵列的圆柱形藻状细胞-软骨细胞结构，这些构造可以压缩五种不同程度的压缩（图1，图3和图4）。气动微通道的高度约为90 μm，PDMS气球直径分别为1.2、1.4、1.6、1.8和2.0 mm。该器件的性能通过具有静态压缩条件和图像处理的共聚焦显...

Watch this Scientific Journal Video about A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression at JoVE.com

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

本文提供了用于软骨细胞压缩的气动微流体装置的制造和特征的详细方法。

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

这种气动活化微流体压缩装置可用于研究生长板软体细胞的三维水凝胶培养物中的力学。微流体器件具有成本和时间上效力，因为它可以同时在小批量样品上生成多个量级的压缩，并且可以与该器件一起应用各种显微成像。该方法也适用于其他细胞类型的力学生物学研究。要设置微通道层，首先将重量比为 10 的 PDMS 与一个固化剂混合 5 分钟。接下来，将模具放在泡沫垫上，将混合物倒入主模具中。将 PDMS 在真空室中脱气 30 分钟，用一块透明薄膜夹夹除气的 PDMS。用玻璃板、泡沫垫和有机玻璃板夹住夹紧夹紧的组件。在 80 摄氏度下治疗 PDMS 6 小时，将 PDMS 层和透明膜与夹层结构隔离。使用等离子清洁剂激活 PDMS 表面和清洁玻璃板 1 分钟，然后将 PDMS 粘合到 80 摄氏度烤箱中的玻璃板上 30 分钟。然后删除透明胶片。对于薄 PDMS 膜的制备，在透明膜上旋转未粘结的 PDMS，每分钟旋转 1，000 次，旋转一分钟，以获得 60 微米厚的 PDMS 层。在使用等离子清洁剂激活玻璃板上的微通道 PDMS 层和薄型 PDMS 层 1 分钟之前，在 80 摄氏度下对旋转涂层 PDMS 进行 20 到 30 分钟的部分固化。然后将薄的 PDMS 膜层粘合到微通道 PDMS 层上，并一夜之间将层放入 80 摄氏度的烤箱中。对于管块制备，将金属管垂直放在培养皿上，然后轻轻地将未加热的 PDMS 倒入培养皿中，直到大约 3/4 的管子被淹没。在真空室中脱气 PDMS 30 分钟后，在 60 摄氏度的烤箱中固化 PDMS 超过 6 小时。在固化过程结束时，切割一块包含一根管子的 PDMS 块，并在细薄的 PDMS 层中打一个孔，用于入口。使用等离子清洁剂激活 PDMS 块和薄型 PDMS 层。一分钟后，将 PDMS 块连接到薄 PDMS 层的进孔，并在 80 摄氏度的烤箱中放置整个驱动单元。对于阿加罗斯凝胶模具制备，在去离子水中混合5%的加糖和200毫摩尔氯化钙，加热阿加罗斯凝胶溶液直到沸腾。将煮熟的阿加罗斯凝胶溶液倒入铝制模具中，将模具与玻璃板夹入。五分钟后，从铝模中去除凝固的加糖凝胶。将藻酸-琼细胞溶液的150微升板放到氨基齐兰化玻璃板上，用琼糖凝胶模具覆盖溶液三分钟。在孵育结束时，使用剃须刀刀片去除过量的藻酸盐凝胶溶液，并从板中取出阿加罗斯凝胶模具。然后将藻酸盐-胆囊细胞结构放入含有50毫摩尔氯化钙和140毫摩尔氯化钠的交联溶液中，在去离子水中放置一分钟。要组装设备，请将四个一毫米厚的 PDMS 分位器定位到驱动单元的薄 PDMS 层的四个角上。用 700 微升的软细胞培养培养剂覆盖薄 PDMS 层的空气室。使用立体微面，将藻酸-夹板结构放在薄的 PDMS 层上，注意将构造与气室对齐，并使用 3D 打印夹住设备。对于设备的驱动，使用一块硅管将空气泵的出水口与电磁阀的入口连接。使用额外的一块管子将电磁阀的插座与组装设备的入口连接。将电磁阀与功能发生器连接，并使用功能发生器生成的 1 赫兹方波操作电磁阀。然后打开空气泵以气动方式驱动设备。微流体软细胞压缩装置包含五到五个圆柱藻酸-软细胞构造数组，这些构造可以通过五个不同规模的压缩进行压缩。在此示例中，最大的 PDMS 气球将凝胶柱的高度压缩了 33.8%，由此产生的凝胶结构压缩应变每 0.2 毫米增量增加约 5%，PDMS 气球直径增加约 5%。在凝胶构造中心附近，通过成像613 by 613 by 40 至 55 微米的细胞，确定软体细胞的压缩变形。这里显示了一个由最大的PDMS气球压缩了16%的软细胞图像。在此图中，可以观察到测量的单元压缩应变值的分布。在此分析中，较大的 PDMS 气球对细胞进行更整体的压缩。综合起来，这些数据表明，藻酸凝胶和软细胞压缩量由PDMS气球的直径在14千帕的恒定压力下控制。为了确保器件性能的可重复性，创建具有恒定厚度和弹性的薄 PDMS 膜至关重要。按照这个程序，可以进行各种生物测定，如免疫荧光和原位杂交。利用这种技术，可以回答有关生长板或关节软骨细胞中的机械转化的许多问题。

Research

JoVE Journal

Bioengineering

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 ...

科研

生物工程

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...