Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.
Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.
In vivo cells are surrounded by a variety of biomolecules including extracellular matrix (ECM), carbohydrates, lipids, and other cells. They functionalize by responding to micro-environmental stimuli such as interactions with ECM and responses to chemical gradients of various growth factors. Traditionally, in vitro cell studies are conducted in cell culture dishes where the consumption of cells and reagents is large and cells grow in a static (non-circulating) environment. Recently, micro-fabricated devices integrated with fluidic components have provided an alternative platform for cell studies in a more controllable way. Such devices are capable of creating a precise micro-environment of chemical and physical stimuli while minimizing the consumption of cells and reagents. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates 1-3. PDMS-based devices are transparent, biocompatible, and permeable to gases, making them suitable for long-term cell culture and studies. PMMA and PET substrates are cheap and easy to be processed using laser ablation and writing.
Microfluidic devices should provide cells with a stable and controllable micro-environment where cells are subject to different chemical and physical stimuli. For example, microfluidic chips are used to study chemotaxis of cells. Instead of traditional methods that employ Boyden chamber and capillary 4,5 these miniaturized fluidic devices can generate precise chemical gradients for studying cells' behaviors 1,6,7. Another example is to study cells' directional migration under electric fields (EFs), a phenomenon named electrotaxis. Electrotactic behaviors of cells were reported to be related to nerve regeneration 8, embryonic development 9, and wound healing 10,11. And many studies have been performed to investigate the electrotaxis of various cell types including cancer cells 12,13, lymphocytes 14,15, leukemia cells 11, and stem cells 16. Conventionally, Petri dishes and cover glasses are used to construct electrotactic chambers for generating EFs 17. Such simple setups pose problems of medium evaporation and imprecise EFs, but they can be overcome by microfluidic devices of enclosed, well-defined fluidic channels 12,18,19.
To systematically study cellular responses under precise, controllable chemical and electrical stimuli, it would be of great use to develop microfluidic devices capable of providing cells with multiple stimuli at the same time. For example, Li et al. reported a PDMS-based microfluidic device for creating single or coexisting chemical gradients and EFs 20. Kao et al. developed a similar microfluidic chip to modulate the chemotaxis of lung cancer cells by EFs 6. Moreover, to increase the throughput, Hou et al. designed and fabricated a PMMA-based multichannel-dual-electric-field chip to provide cells with 8 different combined stimuli, being (2 EF strengths x 4 chemical concentrations)21. To further increase the throughout and add the shear stress stimulus, we developed two PMMA-based microfluidic devices for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.
Reported by Lo et al. 22,23, these devices contain five independent cell culture channels subject to continuous fluidic flowing, mimicking the in vivo circulatory system. In the first chip (the chemical-shear stress chip or the CSS chip), five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 are generated in the culture regions, and a shear stress gradient is produced inside each of the five culture areas. In the second chip (the chemical-electric field chip or the CEF chip), by using one single set of electrodes and 2 syringe pumps, five EF strengths are generated in addition to five different chemical concentrations within these culture areas. Numerical calculations and simulations are performed to better design and operate these chips, and lung cancer cells cultured inside these devices are subject to single or coexisting stimuli for observing their responses with respect to the production of reactive oxygen species (ROS), the migration rate, and the migration direction. These chips are demonstrated to be time-saving, high-throughput and reliable devices for investigating how cells respond to various micro-environmental stimuli.
1. Chip Design and Fabrication
2. Chip Assembly
3. Cell Preparation and Experimental Setup
NOTE: Pre-warm 1x PBS, culture medium (DMEM plus FBS), and trypsin in a 37 °C water bath before usage.
4. Experimental Setup
5. Calculations of Chemical Concentrations, Shear Stresses, and Electric Fields
6. Data Analysis
Note: Data analysis is performed using the ImageJ software.
The Chemical-shear Stress (CSS) Chip
The CSS chip is made of three PMMA sheets, each of thickness 1 mm, attached together via two double-sided tapes, each of thickness 0.07 mm (Figure 1A and 1B). The "Christmas tree" structure generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the five culture areas. By designing the culture area as a triangle, a shear stress gradient, with a magnitude related the volume flow rate, the fluidic viscosity, and the dimension of the fluidic channel, is created within each of the areas. This chip is then attached, via another 0.07 mm thick double-sided tape, to a Petri dish for culturing lung cancer CL1-5 cells and the production of ROS was observed in response to different chemical concentrations and shear stresses. The production of ROS in lung cancer cells is highly related to lung cancer metastasis and development, and certain chemicals and shear stress were demonstrated to be involved in ROS generation 23.
First, to study the effect of H2O2, a chemical stimulus, on the production of ROS, cells were incubated with continuous flowing of H2O2 solutions at 0, 25, 100, 175, and 200 µM. As shown in Figure 1C, the fluorescent intensity increased as the concentration of H2O2 increased, indicating that H2O2 stimulated the production of ROS. Next, to investigate the effect of shear stress on the production of ROS, cells were exposed to a shear stress gradient of 0.0048 Pa to 0.0192 Pa. Figure 1D shows that the fluorescent intensity increased as the shear stress increased (the shear stresses were the highest and the lowest in the Front and Back areas, respectively), suggesting that higher shear stress induced more ROS production. Also, this CSS chip was used to study the production of ROS in response to different concentrations of α-tocopherol, an antioxidant of a form of vitamin E. Cells were stimulated by shear stress plus α-tocopherol of 0, 3.2, 12.5, 21.9, and 25 µg/ml. As shown in Figure 1E, for α-tocopherol concentrations lower than 21.9 µg/ml, the fluorescent intensity decreased as the concentration increased, indicating the effect of α-tocopherol in reducing the production of ROS. However, as the concentration increased to 25 µg/ml, the mean intensity also increased, suggesting that this high concentration of α-tocopherol did not eliminate much ROS compared to lower concentrations.
The Chemical-electric Field (CEF) Chip
The CEF chip is made of a 1 mm-thick PMMA and a 0.22-mm double-sided tape with fluidic channels patterned on it (Figure 2A and 2B). Similarly, the "Christmas tree" structure creates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the five culture areas. In addition, these five parallel areas are connected perpendicularly to form a fluidic path analogous to an electric circuit. The electric field generated within a culture area is related to the conductivity of the fluid, the cross-sectional area of the channel, and the direct current (dc) passing through the area. According to Kirchhoff's voltage law and Ohm's law, the currents in the five culture areas are 0.49I, 0.25I, 0.13I, 0.08I, and 0.05I, respectively, where I is the applied dc (Figure 2C). This chip is attached, via the 0.22 mm thick double-sided tape, to a Petri dish for culturing lung cancer CL1-5 cells to observe cell migration and the production of ROS in response to different chemical concentrations and electric fields.
First, this chip was used to study the production of ROS in response to different concentrations of honokiol, different strengths of EFs, and combined treatments of both. Honokiol, a small-molecule polyphenol isolated from the genus Magnolia, was found to have antiangiogenic, anti-inflammatory, and antitumor properties in preclinical studies 28. As shown in Figure 2D, the ROS produced was almost the same for EFs lower than 67 mV/mm, but was increased with increasing EFs above this value. Figure 2E shows that under combined treatments of electric fields and honokiol, the ROS level stayed almost the same, indicating that honokiol inhibited the production of exogenous ROS (i.e., ROS related to EF stimulus), especially under higher EFs. The cell migration under single or coexisting chemical/electrical stimuli was also investigated using this CEF chip. As seen in Figure 2F, without the addition of honokiol, the migration rate increased as the EF strength increased. After adding honokiol of different concentrations, the migration rate decreased in general, suggesting that honokiol reduced cell migration possibly via inhibiting the production of ROS. The migration directedness is shown in Figure 2G. In the presence of EF only, lung cancer cells showed prominent directional migration toward the anode (with negative values). After adding honokiol of different concentrations, there was a slight decrease in migration directedness for all EF-stimulated areas.
Figure 1: The Chemical-shear Stress (CSS) Chip Used to Study the Effects of Chemicals and Shear Stresses on Lung Cancer Cells.23
(A) Three layers of PMMA substrates are bound together via two double-sided tapes to form the CSS chip. (B) The integrated CSS chip. (C) Top: Fluorescent and bright-field images of CL 1-5 cells after being treated with different concentrations of H2O2. Scale bar = 50 µm. Bottom: Mean fluorescent intensity with SEM plotted at different concentrations of H2O2. (D) Top: Fluorescent images of CL 1-5 cells under different shear stresses. Scale bar = 50 µm. Bottom: Mean fluorescent intensity with SEM plotted at different shear stresses. (E) Top: Fluorescent and bright-field images of CL 1-5 cells after being stimulated with shear stress with different concentrations of α-tocopherol. Scale bar = 50 µm. Bottom: Mean fluorescent intensity with SEM plotted at different concentrations of α-tocopherol. Data presented here was originally published in reference 23. Please click here to view a larger version of this figure.
Figure 2: The Chemical-electric field (CEF) Chip Used to Study the Effects of Chemicals and Electric Fields on Lung Cancer Cells 22.
(A) One layer of PMMA is bound onto a culture dish via a double-sided tape to form the CEF chip. (B) The fluidic pattern of the CSS chip. (C) The equivalent electrical circuit of the microfluidic chip. (D) Left: Fluorescent and bright-field images of CL 1-5 cells after being treated with different strengths of EFs. Scale bar = 50 µm. Right: Mean fluorescent intensity with SEM plotted at different strengths of EFs. (E) Left: Fluorescent and bright-field images of CL 1-5 cells after being treated with combined honokiol and EFs. Scale bar = 50 µm. Right: Mean fluorescent intensity with SEM plotted at combined honokiol and EFs. (F) The migration rates of CL1-5 cells after being treated with EFs only (marked as control) and combined honokiol and EFs (marked as honokiol). (G) The migration directedness of CL1-5 cells after being treated with EFs only (marked as control) and combined honokiol and EFs (marked as honokiol). Data presented here was originally published in reference 22. Please click here to view a larger version of this figure.
PMMA-based chips are fabricated using laser ablation and writing which are cheaper and easier methods when compared to PDMS-based chips which require more complicated soft lithography. After designing a microfluidic chip, the fabrication and assembly can be done within just 5 min. There are some critical steps that attention should be paid to in performing the experiment. The first is the "assembling" issue. The adaptors should be glued properly to the top-most layer of the chip. Glue could leak into the fluidic channels if too much is applied, and liquid could leak out of the chip if too little glue is used. Also, in assembling the PMMA substrates and the double-sided tapes, it is important to apply pressure to press the whole chip tightly to prevent any liquid leakage. The second is the "bubble" issue. To remove bubbles, 1x PBS is continuously flowed into the fluidic channel for 1 or 2 min at a flow rate of 20 µl/min and then the chip is placed inside an incubator overnight under 5% CO2 at 37 °C. If there are bubbles remaining within the fluidic channels, the flow will be non-uniform, causing nonhomogeneous chemical concentration and EF distribution. The third is the "cell loading" issue. The number of cells loaded into the culture areas should be well controlled. If the cell number is too low, many experimental runs should be conducted to collect enough data for statistical purpose. If the cell number is too high, it will be difficult to distinguish individual cells and quantify their migration.
In the CSS chip, a "Christmas tree" structure combined with a triangular culture area generates different chemical concentrations in five areas, each having a shear stress gradient. Fluidic shear stresses were known to affect the attachment, the morphology, the migration, and the activity of cells. Shear stresses of as low as 0.25-0.6 Pa could interface cell attachment, and even higher values of stresses (0.5 -10 Pa) were reported to remove adherent cells 29. Laminar shear stresses ranging from 0.8 to 1.5 Pa induced cell alignment in the direction of flow 30, and even lower values (0.1-1 Pa) were known to affect cellular morphology and permeability 29. Moreover, smooth muscle cells subjected to shear stresses of 2-120 Pa experienced a decline in cell number 31. Lu et al. reported the design and construction of microfluidic shear devices for quantitative analysis of cell adhesion 27. The shear stress within the fluidic channel was modified by changing the dimensions of the channel. Chin et al. described a hemodynamic Lab-on-a-chip system for controlling the flow rate of the culture medium in the micro-channel to mimic the flow profile of the blood in the vessel 32. The shear stress within the fluidic channel was modified by changing the flow rate of the medium. In these two devices, even though a shear stress of any values could be generated, only one single value was available within one culture area at one time. By comparison, the present CSS chip has the advantage of providing a shear stress gradient in one triangular area, increasing the experimental throughput especially for a screening purpose.
In the CEF chip, the culture areas of the "Christmas tree" structure are perpendicularly connected to form a fluidic path analogous to an electric circuit. At one applied direct current, five EF strengths are generated inside the culture areas with each having a flow of a specific chemical concentration. Conventionally, electrotactic experiments were performed on Petri dishes where problems of imprecise EFs, medium evaporation, large cell/reagent consumption, and increased Joule heating could occur. Enclosed, miniature microfluidic devices efficiently overcome these drawbacks. For example, to study the electrotaxis of human blood memory T cells, Lin et al. reported a plastic microfluidic device containing two identical, side-by-side micro-channels, two modified pipette tips served as medium reservoirs, and two platinum electrodes for EF application 15. To increase the throughput, PMMA-based electrotactic chips were fabricated to provide multiple EF strengths in one single device 12,33. Also, microfluidic devices capable of generating controllable chemical and electrical stimuli simultaneously are of high interest. Li et al. fabricated a PDMS-based microfluidic device to generate single or coexisting chemical gradients/EFs for investigating the chemotactic or electrotactic migration of T cells 14. Kao et al. employed a similar microfluidic device to modulate the chemotaxis of lung cancer cells by using EFs 12. In these two devices, a Y-shape structure was applied to create a stable concentration gradient in one single applied EF. Hou et al. reported a multichannel-dual-electric-field chip to study the concurrent effect of chemicals and EFs on lung cancer cells21. This device was able to provide 8 combinations of electrical/chemical stimuli (2 electrical x 4 chemical) in one experiment. Although the throughput increased, this chip's capacity was limited to (1) only one EF strength (plus one zero as a control), and (2) four syringe pumps are required for pumping chemicals into four independent channels. By comparison, the present CEF chip has the advantage of generating five EF strengths together with five chemical concentrations via one single set of electrodes and 2 syringe pumps.
Even though these chips are easy to be fabricated, they have some limitations. First, only five "specific" concentrations and five "specific" EF strengths can be generated. Second, the width of the fluidic channel cannot be less than 1 mm due to the focusing of the CO2 laser beam. However, by precisely controlling the chemical concentration in the inlet and the electric current passing through the fluidic channel, any concentrations and EF strengths can be attained in the culture areas. In conclusion, the present CSS and CEF chips are capable of providing cells with controllable single or coexisting chemical/electrical/shear stress stimuli. In one single CSS chip, five different chemical concentrations in combination with a shear stress gradient can be generated. In addition, in one single CEF chip, five different chemical concentrations in combination with five EF strengths can be created. By increasing the branches of the "Christmas tree" structure, the throughput of these chips can be further increased. These chips are demonstrated to be an easy, time-saving, reliable, and high-throughput platform for studies of cellular behavior under chemical/electrical/shear stress stimuli.
The authors have nothing to disclose.
This work was financially supported by the Ministry of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026 (K. Y. Lo), No. MOST 104-2112-M-030-002 (Y. S. Sun), and National Taiwan University Career Development Project (103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material and Advanced Devices, National Taiwan University, for the use of the cell culture room.
Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11965-092 | Cell culture medium |
Trypsin | Gibco | 25300-054 | detach cell from the dish |
Fetal bovine serum (FBS) | Gibco | 10082147 | Cell culture medium |
10-cm cell culture Petri dish | Nunc | 150350 | Cell culture |
Bright-Line Hemacytometer | Sigma | Z359629 | Cell Counting Equipment |
PMMA | Customized | Customized | Microfluidic chip |
Adaptor | Customized | Customized | Microfluidic chip |
0.07/0.22 mm double-sided tape | 3M | 8018/9088 | Microfluidic chip |
Low melting point agarose | Sigma | A9414 | Salt bridge |
2'-7'-dichlorodihydrofluoresce diacetate | Sigma | D6883 | Intracellular ROS measurement |
Indium tin oxide (ITO) glass | Merck | 300739 | Heater |
Proportional-integral-derivative controller | JETEC Electronics Co. | TTM-J4-R-AB | Temperature controller |
Thermal coupler | TECPEL | TPK-02A | Temperature controller |
CO2 laser scriber | Laser Tools & Technics Corp. | ILS2 | Microfluidic chip fabrication |
Syringe pumps | New Era | NE-300 | Pumping medium and chemicals into the chip |
Power supply | Major Science | MP-300V | Supplying direct currents |
Inverted microscope | Olympus | CKX41 | Monitoring cell migration |
Inverted fluorescent microscope | Nikon | TS-100 | Monitoring cell migartion and fluorescencent signals |
DSLR camera | Canon | 60D | Recording bright-field images |
CCD camera | Nikon | DS-Qi1 | Recording fluorescent images |
super glue | 3M Scotch | 7004 | Attaching adaptors to PMMA substrates |
AutoCAD | Autodesk Inc. | Designing microfluidic chips | |
DMSO | Sigma | D8418 | Dissolving DCFDA |
ImgeJ | National Institutes of Health | Quantifying fluorescent intensities and cell migration |