微流体平台，用于用动态压缩刺激软骨细胞
1Department of Genetics, Cell Biology and Anatomy,University of Nebraska Medical Center, 2Department of Physiology and Pharmacology,Karolinska Institutet, 3Department of Mechanical and Materials Engineering,University of Nebraska-Lincoln, 4Nebraska Center for Materials and Nanoscience,University of Nebraska-Lincoln
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 玻璃处理注：为步骤 1.1 佩戴面罩、手套和实验室外套。 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?…
Representative Results
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…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| (3-Aminopropyl)triethoxysilane (ATPES) | Sigma-Aldrich | 741442-100ML | |
| (Tridecafluoro-1, 1, 2, 2-Tetrahydrooctyl)-1-Trichlorosilane | United Chemical Technologies | T2492-KG | |
| Acrylic sheet | McMaster-Carr | 8560K354 | |
| Air pump | Schwarzer Precision | SP 500 EC-LC4.5V DC | 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. |
| Alginate powder | FMC Corporation | Pronova UP MVG | |
| Barb Straight Connectors (Metal tube) | Pneumadyne | EB40-250 | |
| Calcein AM | Invitrogen | C3100MP | |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11960-044 | |
| Dyed red aqueous fluorescent particles | Thermo Fisher Scientific | R0100 | |
| EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) | Thermo Fisher Scientific | 22980 | |
| Foam pad | GRAINGER | Item # 5GCE8 | |
| Function / Arbitrary Waveform Generator | Keysight Technologies | 33210A | |
| Hydrochloric acid | Fisher Chemical | A144-500 | |
| Hydrogen peroxide | Fisher BioReagents | BP2633500 | |
| Isopropyl alcohol | BDH1174-4LP | VWR | |
| Microscope slides | Thermo Fisher Scientific | 22-267-013 | |
| Plasma cleaner | Harrick Plasma | PDC-001 | |
| Polydimethylsiloxane (PDMS) | Dow Corning | 184 SIL ELAST KIT 0.5KG | |
| Power supply | Keysight Technologies | E3630A | |
| SeaKem LE Agarose | Lonza | 50004 | |
| Sodium hydroxide | Fisher Chemical | S318-1 | |
| Solenoid manifold | Pneumadyne | MSV10-1 | |
| Solenoid valve | Pneumadyne | S10MM-30-12-3 | |
| Spin coater | Laurell Technologies | WS-650Mz-23NPPB | |
| SU8 Developer | MicroChem Corp. | Y020100 4000L1PE | |
| SU8-100 | MicroChem Corp. | Y131273 0500L1GL | |
| SU8-5 | MicroChem Corp. | Y131252 0500L1GL | |
| Sulfo-NHS (N-hydroxysulfosuccinimide) | Thermo Fisher Scientific | 24510 | |
| Sulfuric acid | EMD Millipore | MSX12445 |
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Citazione di questo articolo
Lee, D., Erickson, A., Dudley, A. T., Ryu, S. A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression. J. Vis. Exp. (151), e59676, doi:10.3791/59676 (2019).