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.
This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1α) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.
The spatial distribution of soluble factors and oxygen tension can regulate a number of important cellular functions in vivo1,2,3,4. In order to better investigate their effect on cells, an in vitro cell culture platform capable of stably generating chemical and oxygen gradients is highly desired. Various soluble factors play key roles in biological activities and affect cellular behavior. Recently, due to the advancement of microfluidic techniques, a number of microfluidic devices capable of stably generating chemical gradients have been developed to study cell migration5. Furthermore, several studies have also revealed the necessity of oxygen tension for in vitro cell cultures6,7,8. However, the control of oxygen tension for cell culture mainly relies on direct chemical addition for oxygen scavenging or cell incubators with pressurized gas cylinders. Direct chemical addition alters the cell culture medium and affects the cellular responses. Oxygen control incubators require special incubator design, precise gas flow control, and a large volume of nitrogen gas in order to achieve hypoxia conditions. Moreover, it is infeasible to control the spatial distribution of oxygen using this setup, which retards the cellular behavior study under various oxygen tensions and gradients. To overcome these limitations, a number of microfluidic devices have been developed to generate oxygen gradients for cell culture applications9. However, most of them are operated using pressurized gases, which may cause evaporation and bubble generation problems. Therefore, they often require sophisticated instrumentation and may not be reliable for long-term cell culture studies.
To overcome the challenges and to further study the interactions between chemical and oxygen gradients for cell migration, we developed a microfluidic cell culture device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film10. The device is composed of two microfluidic channel layers separated by a PDMS membrane. The top layer is a PDMS-PC layer for oxygen gradient generation; the bottom layer is made of PDMS for chemical gradient generation and cell culture. The device can simultaneously generate perpendicular chemical and oxygen gradients without using gas cylinders and sophisticated flow control systems. In the device, PDMS provides great optical transparency, gas permeability, and biological compatibility for cell culture and imaging. The embedded PC film serves as a gas diffusion barrier for efficient oxygen tension control. In the microfluidic channel, we used a series of serpentine-shaped channels to generate chemical gradients. The design has been broadly exploited to generate chemical gradients in microfluidic devices for various applications due to its reliability and easy experimental setup. Furthermore, the chemical gradient profiles can be designed by varying the channel geometries beforehand with numerical simulation. For oxygen gradient generation, we took advantage of the spatially confined chemical reaction method that was previously developed in our lab10,11,12. The oxygen can be scavenged from the designated areas without nitrogen purging. For practical usage in biological labs, the entire experimental setup is compatible with conventional cell culture incubators. By integrating these methods, we can simultaneously generate stable chemical and oxygen gradients without bulk gas cylinders and sophisticated instrumentation in order to study cell migration.
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.
The authors have nothing to disclose.
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.
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 |