细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置
Summary
The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.
Abstract
本文报道具有嵌入式聚碳酸酯（PC）薄膜研究根据化学和氧梯度的组合细胞迁移制成聚二甲基硅氧烷（PDMS）的微流体装置。化学和氧梯度能极大地影响体内细胞迁移;然而，由于技术限制，很少研究已经进行，调查它们的体外作用。在此研究开发的设备需要一系列蛇形的通道，以产生所希望的化学梯度的优势，并利用氧梯度产生在空间上限于化学反应方法。化学和氧梯度的方向是彼此垂直的，以使直接的迁移的结果的解释。为了有效地生成具有最小的化学品消耗的氧梯度，嵌入式PC薄膜被用作气体扩散阻挡层。所开发的微流体装置可以通过注射泵被致动，并在细胞迁移实验放入常规的细胞培养器，以允许设置简化和优化的细胞培养条件。在细胞实验中，我们使用的设备来研究adenocarcinomic人肺泡基底上皮细胞，A549细胞迁移，下的趋化因子的组合（基质细胞衍生因子，SDF-1α）和氧梯度。实验结果表明，该装置能够稳定地产生垂直的趋化因子和氧梯度和与细胞相容。迁移研究结果表明，氧梯度可能在引导细胞迁移下梯度的组合不能从那些在单个梯度预测了至关重要的作用，以及细胞的行为。该设备为研究人员提供研究在细胞培养化学和氧梯度之间的相互作用一个强大而实用的工具，它可以促进更多的体内样microenvi更好的细胞迁移的研究ronments。
Introduction
的可溶性因子和氧气张力的空间分布可以在体内 1，2，3，4调节一些重要的细胞功能。为了更好地研究其对细胞的影响，体外细胞培养物能够稳定地产生化学和氧梯度平台是高度期望的。各种可溶性因子的生物活性起着关键的作用，影响细胞行为。最近，由于微流体技术的进步，许多能够稳定地产生化学梯度的微流体装置已被开发，研究细胞迁移5。此外，一些研究也揭示了氧分压的体外细胞培养物6，7，8的必要性。然而，氧张力的用于细胞培养的控制主要依赖于直接化学添加用于与加压气瓶的除氧或细胞孵化。直接化学除了改变细胞培养基，影响细胞应答。氧气控制孵化需要特殊培养箱设计，精确的气体流量控制，并以实现低氧条件大量氮气。此外，它是不可行的控制使用此设置，这阻碍了在各种氧张力和渐变的细胞行为研究氧气的空间分布。为了克服这些限制，一些微流体装置已被开发，以产生用于细胞培养应用9氧气梯度。然而，其中大部分是使用加压气体，这会引起蒸发和气泡产生的问题进行操作。因此，它们经常需要复杂的仪器和可能不可靠的长期细胞培养studie秒。
为了克服的挑战，并进一步研究化学和氧梯度之间的相互作用对细胞迁移，我们开发了具有嵌入式聚碳酸酯（PC）薄膜10制成聚二甲基硅氧烷（PDMS）的微流体细胞培养设备。该装置是由通过一个PDMS膜分离的两个微流体通道的层。顶层为氧梯度产生一个PDMS-PC层;底部层是由PDMS制成用于化学梯度的产生和细胞培养的。该设备可以同时产生垂直化学和氧梯度，而不使用气瓶和精密的流量控制系统。在该装置中，PDMS提供了极大的光学透明性，透气性，以及用于细胞培养和成像生物兼容性。嵌入式PC薄膜用作有效的氧张力控制的气体扩散阻挡层。在微流体通道中，我们使用一系列蛇形通道的s到产生化学梯度。设计已经被广泛地利用以产生用于各种用途的微流体装置化学梯度由于其可靠性和容易的实验装置。此外，化学梯度分布可以通过数值模拟事先改变通道的几何形状进行设计。氧梯度产生，我们采取了先前在我们实验室10，11，12开发的空间上的限制化学反应的方法的优点。氧气可以从指定区域无氮气吹扫被清理。用于生物实验室实际使用中，整个实验装置与传统的细胞培养孵化器兼容。通过整合这些方法，我们可以同时产生无散装气瓶和复杂仪器稳定的化学和氧梯度，以便研究细胞迁移。
Protocol
Representative Results
Discussion
The most critical steps to fabricate the PDMS microfluidic device with an embedded PC thin film are: (1) expelling all the bubbles when inserting the PC film into the PDMS pre-polymer while fabricating the PDMS-PC top layer and (2) making sure all the PDMS curing processes are performed on a well-leveled horizontal plane. For cell migration experiments, the most critical steps are: (1) eliminating the bubbles within the microfluidic device, tubing, and syringe pumps during the experiments; (2) ensuring that the microfluidic device is placed on a well-leveled horizontal plane during live cell imaging for the cell migration observation; and (3) keeping the device moisturized by adding ddH2O to the Petri dish during the experiments and making sure that the water is not dried out.
In order to successfully fabricate the PDMS-PC hybrid microfluidic device without delamination, it is critical to remove all bubbles during the PC film insertion. The PC film can be slowly inserted from an angle (about 15 to 30 degrees away from the PDMS pre-polymer surface) to prevent bubbles generation during the insertion of the PC film into the PDMS pre-polymer. If necessary, the entire PDMS pre-polymer with the embedded PC film can be placed in the desiccator connected to the vacuum pump for 10 min to expel the trapped bubbles. If the PC film floats up after the vacuum process, a pipette tip can be used to press the PC film down onto the cured PDMS layer. Repeat the vacuum and press processes if necessary.
For the cell experiments, a lack of air bubbles is critical for the microfluidic cell culture. Make sure that no air bubbles are introduced into the entire microfluidic setup (including syringe pumps, tubing, and the microfluidic device) when making connections. If air bubbles are created within the microfluidic setup due to the decrease of gas solubility under the elevated temperature of the experiments inside the incubator, all the experimental components (including the syringes and tubing) and reagents (including the growth medium, pyrogallol, and NaOH) can be placed into the incubator beforehand (at least 20 min prior to usage) to minimize the temperature variation. Syringe pumps often generate heat from the operation of the motors within the pumps. It is usually acceptable to operate syringe pumps inside incubators; however, do check the incubator temperature during the experiments. If the temperature elevates during the experiments, additional cooling procedures need to be carried out. Several feasible cooling methods can be employed, such as placing a box of ice into the incubator, reducing the number of syringe pumps placed inside the incubator, or using an incubator with a force cooling system.
The PDMS-PC microfluidic cell culture device developed in this paper is capable of reliably generating perpendicular chemical and oxygen gradients for cell migration studies. The limitation of the developed device is that the generated oxygen gradient profiles depend on the balance between oxygen flux, driven by chemical reaction scavenging, and oxygen diffusion from the ambient atmosphere, through the device, and into the medium. As a result, the oxygen gradient profiles cannot be arbitrarily controlled inside the device. Compared to existing microfluidic cell culture platforms, the device developed in this paper is the first one capable of performing cell culture studies under combinations of chemical and oxygen gradients. The entire device can be fabricated using the conventional soft lithography replica molding process, without tedious alignment and expensive instrumentation. The gradients can be numerically simulated and experimentally characterized to provide more physiological microenvironment-like conditions for in vitro cell studies. By using a spatially confined chemical reaction method with a PC film as a gas diffusion barrier, the oxygen gradient can be generated without using pressurized gas cylinders and sophisticated gas flow control units. In addition, the setup requires only small amounts of chemicals (less than 10 ml per day) to maintain the oxygen gradients. Since the oxygen tension control is confined locally around the microfluidic channel, and does not disturb the ambient oxygen concentration, the entire setup can be placed inside a conventional cell culture incubator without additional temperature, humidity, and CO2 control instrumentation. As a result, the developed device has great potential to be practically used in biological labs.
Due to technical limitations, cellular behaviors under oxygen tensions have seldom been studied in the existing literature. With the help of the device developed in this paper, cell culture under oxygen gradients can be performed in a facile manner that greatly promotes cell studies under oxygen gradients. Furthermore, a similar operation principle can be applied to generate other physiologically relevant gaseous gradients, such as carbon dioxide and nitric oxide, for in vitro cell culture studies17. These capabilities demonstrate that the PDMS-PC microfluidic device shows great potential for various cell culture applications, including drug testing and cell proliferation and migration assays, to advance in vitro cell culture studies.
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This paper is based on work supported by the National Health Research Institutes (NHRI) in Taiwan under the Innovative Research Grant (IRG) (EX105-10523EI), the Taiwan Ministry of Science and Technology (MOST 103-2221-E-001-001-MY2, 104-2221-E-001-015-MY3, 105-2221-E-001-002-MY2), the Academia Sinica Thematic Project (AS-105-TP-A06), and the Research Program in Nanoscience and Nanotechnology. The authors would like to thank Ms. Rachel A. Lucas for proofreading the manuscript.
Materials
| 1 ml Syringe | Becton-Dickinson, Franklin Lakes, NJ | 302104 | |
| 1.5 ml Microcentrifuge Tube | Smartgene | 6011-000 | |
| 10 ml Syringe | Becton-Dickinson, Franklin Lakes, NJ | 302151 | |
| 15 ml Centrifuge Tube | ThermoFisher Scientific,Waltham, MA | Falcon 352096 | |
| 150 mm Petri-Dish | Dogger Science | DP-43151 | |
| 1H,1H,2H,2H-Perfluorooctyltrichlorosilane | Alfa Aesar, Ward Hill, MA | 78560-45-9 | |
| 3 ml Syringe | Becton-Dickinson, Franklin Lakes, NJ | 302118 | |
| 4'' Silicon Dummy Wafer | Wollemi Technical, Taoyuan, Taiwan | ||
| Acetone | ECHO Chemical, Miaoli, Taiwan | AH3102-000000-72EC | |
| AG Double Expose Mask Aligner | M&R Nano Technology, Taoyuan, Taiwan | AG500-4D-D-V-S-H | |
| Antibiotic-Antimyotic solution | ThermoFisher Scientific,Waltham, MA | GIBCO 15240-062 | |
| Biopsy punch | Miltex, Plainsboro, NJ | 33-31 | |
| Blunt needle | JensenGlobal, Santa Barbara, CA | Gauge 14 | |
| Bright-Line Hemocytometer | Sigma-Aldrich, St. Louis, MO | Z359629 | for cell counting |
| Buffered Oxide Etch | ECHO Chemical, Miaoli, Taiwan | PH3101-000000-72EC | |
| Cell Culture Incubator | Caron, Marietta, OH | 6016-1 | |
| COMSOL Multiphysics | COMSOL, Burlington, MA | Ver. 4.3b | for numerical simulation of chemical gradients in the device |
| D-PBS | ThermoFisher Scientific,Waltham, MA | GIBCO 14190-144 | |
| Desicattor | A-VAC Industries, Anaheim, CA | 35.10001.01 | |
| DMEM/ F12+GlutaMax-1 | ThermoFisher Scientific,Waltham, MA | GIBCO 10565-018 | |
| Fetal Bovine Serum | ThermoFisher Scientific,Waltham, MA | GIBCO 10082 | |
| Fibronectin from Human Plasma | Sigma-Aldrich, St. Louis, MO | F2006 | |
| Inverted Fluorescence Microscope | Leica Microsystems, Wetzlar, Germany | DMIL LED | |
| Isopropyl Alcohol (IPA) | ECHO Chemical, Miaoli, Taiwan | CMOS112-00000-72EC | |
| JuLi Smart Fluorescence Cell Imager | NanoEnTek, Seoul, Korea | DBJ01B | |
| Mechanical Convention Oven | ThermoFisher Scientific,Waltham, MA | Lindberg Blue M MO1450C | |
| NaOH | Showa Chemical Industry, Tokyo, Japan | 1943-0150 | |
| Plasma tretment system | Nordson MARCH, Concord CA | PX-250 | for oxygen plasma surface treatment |
| Polycarbonate (PC) film | Quantum Beam Technologies, Tainan Taiwan | ||
| Polydimehtylsiloxane (PDMS) | Dow Corning, Midland, MI | SYLGARD 184 | |
| Pyrogallol | Alfa Aesar, Ward Hill, MA | A13405 | |
| Removable Adhesive Putty | 3M | 860 | |
| Sorvall Legend Mach 1.6R Tabletop Centrifuge | ThermoFisher Scientific,Waltham, MA | ||
| Spin Coater | ELS Technology, Hsinchu, Taiwan | ELS 306MA | |
| SU-8 2050 | MicroChem, Westborough, MA | SU-8 2050 | |
| SU-8 Developer | MicroChem, Westborough, MA | Y020100 | |
| Surgical blade | Feather, Osaka, Japan | 5005093 | for PDMS cutting |
| Syringe Pump | Chemyx, Houston, TX | Fusion 400 | |
| T75 Cell Culture Flask | ThermoFisher Scientific,Waltham, MA | Nunc 156367 | |
| Trypan Blue Solution, 0.4% | ThermoFisher Scientific,Waltham, MA | 15250061 | |
| Trypsin-EDTA | ThermoFisher Scientific,Waltham, MA | GIBCO 25200 | |
| Tygon PTFE Tubing | Saint-Gobain Performance Plastics, Akron, OH | ||
| Tygon Tubing | Saint-Gobain Performance Plastics, Akron, OH | 621 |
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