Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

1. Chip Design and Fabrication

1. Draw patterns to be ablated on PMMA substrates and double-side tapes using commercial software ²⁴.
   1. To study the effects of chemical concentrations and shear stresses, draw a "Christmas tree" pattern with a varying width at its end in each of the five culture areas (Figure 1A and 1B).
   2. To study the effects of chemical concentrations and electric fields, draw a "Christmas tree" pattern with two more fluidic channels for salt bridges (Figure 2A and 2B).
2. Scribe an individual pattern on a PMMA sheet or a double-side tape by loading the corresponding file into the CO₂ laser scriber.
   1. Turn on the laser scriber, and check its connection to the computer. Open the pattern to be ablated using the commercial software.
   2. Place a PMMA sheet or a double-sided tape on top of the stage of the scriber. Adjust the focus of the CO₂ laser if necessary using the calibration bar and the visible He-Ne laser.
   3. Load the pattern into the scriber for ablation on the PMMA sheet or the tape.
   4. Pick up the patterned sheet or tape, remove unwanted pieces, and clean the surface with nitrogen blowing.
      NOTE: The thickness of the PMMA sheet is 1 mm, and that of the double-sided tape is 0.07 mm or 0.22 mm.

2. Chip Assembly

1. With super glue, attach acrylic adaptors (length x width x height = 10 mm x 10 mm x 5 mm, with screw threads in the middle) to the top-most layer of the chip by aligning the screw threads and the holes on the top-most layer. These adaptors serve as medium inlets/outlets and salt bridge connectors.
2. Assemble the microfluidic chip inside a laminar flow hood.
3. Assemble the chemical-shear stress microfluidic chip (CSS chip).
   1. Attach 3 sheets of scribed PMMA sheets using 2 pieces of scribed double-sided tape. (Figure 1A and 1B).
   2. Add one more piece of the 0.07 mm thick double sided tape to the bottom of the chip and attach the chip to a 10 cm diameter Petri dish.
   3. Put the assembly chip in the vacuum chamber for overnight.
4. To assemble the chemical-electric field chip (CEF chip), attach the PMMA sheet to a double-sided tape of thickness = 0.22 mm (Figure 2A and 2B).
   1. Attach the scribed PMMA sheet and double-sided tape to a 10 cm diameter Petri dish using this same piece of tape.
5. Leave the assembled chip inside the hood and expose it to UV for 30 min for sterilization. 
6. Connect the inlets to two 3-ml syringes via plastic tubes and finger-tight nuts. Connect the outlet to a waste tube via a plastic tube and a finger-tight nut.
   NOTE: Autoclave all tubes and nuts at 121 °C for 15 min prior to usage.
7. Slowly depress the plunger on the syringe to prime the fluidic channels with PBS. Push syringes back and forth to remove bubbles.
8. Put the chips inside an incubator overnight at 37 °C under 5% CO₂.
   NOTE: These two steps are aimed to wash the chip and remove any remaining bubbles within the chip.
9. Take the chip out of the incubator.
10. Prepare agar salt bridges for generating electric fields in the CEF chip. 
    1. Dissolve 3% low melting point agarose in 1x phosphate buffered saline (PBS) buffer using a microwave. 
    2. Inject solution-phased agarose into the salt bridge channel and insert the electrodes before solution solidifies.
    3. Place silver/silver-chloride electrodes into the tubular nuts.
11. Connect the syringes to the syringe pumps, and continuously flow the culture medium (Dulbecco’s Modified Eagle’s medium, DMEM) into the fluidic channel for 10 min at a flow rate of 20 μl/min to replace PBS.

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.

1. Plate 2 x 10⁵ lung cancer CL1-5 cells^25,26 in a 10 cm Petri dish supplied with DMEM plus 10% fetal bovine serum (FBS). Incubate the cells inside an incubator under 5% CO₂ at 37 °C until 90% confluence.
2. Aspirate the medium and wash the cells once with pre-warmed 1x PBS. Add 2 ml of 0.05% trypsin buffer to the cells and wait for 2 to 3 min at 37 °C to detach the cells.
3. Transfer the cells to a 15 ml sterile centrifugation tube and add 6 ml of culture medium into the tube. Gently invert the tube for mixing and take out 5 µl of the cell-containing medium for counting the cell number in a hemocytometer.
4. Centrifuge the tube at 300 x g for 5 min. Suspend 10⁶ cells in 1 ml of culture medium and place the cell-containing medium in a 1 ml syringe.
5. Infuse the cell-containing medium into the microfluidic chip from the outlet and make sure that the solution distributes all through the five culture areas. Incubate the chip inside an incubator under 5% CO₂ at 37 °C for 2 hr. 

4. Experimental Setup

1. Take the chip out of the incubator and place it on top of a transparent indium tin oxide (ITO) glass heater.
   NOTE: The ITO glass is connected to a proportional-integral-derivative (PID) controller for maintaining the temperature at 37 ± 0.5 °C via feedback from a thermal coupler clamped tightly between the ITO heater and the chip.
2. Put the chip-heater assembly on top of a motorized XY stage of an inverted microscope for tracking cell migration or a fixed XY stage of an inverted fluorescent microscope for measuring the production of ROS.
3. To generate different chemical concentrations, fill two syringes with 5 ml of chemical solutions of relative concentrations 0 and 1 (dissolved in culture medium) and pump them into the chip at desired flow rates: in the CSS chip, at a flow rate of 0.3 ml/min for 1 hr; in the CEF chip, at a flow rate of 20 µl/min for the first 20 min and a flow rate of 20 µl/hr for another 120 min (total time = 2 hr).
4. In the CEF chip, for tracking cell migration, program the XY stage of the microscope to repeat taking photos, via a digital single-lens reflex (DSLR) camera, of certain field of views (FOVs) within culture areas every 15 min for 2 hr.
5. For measuring the production of ROS, use the fluorescence-based indicator 2′-7′-dichlorodihydrofluoresce diacetate (2′-7′-DCFDA).
   1. Prepare the stock of 2′-7′-DCFDA at 10 mM in molecular biology grade dimethyl sulfoxide (DMSO). Dilute DCFDA in DMEM only without serum (5 µM in DMEM). The potential deacetylase could increase the background signals and decrease the signals in cells.
   2. After 1 hr of shear stress stimulus in the CSS chip or after 2 hr of EF stimulus in the CEF chip, pump 2′-7′-DCFDA (5 µM in DMEM) into the chip at a flow rate of 20 µl/min for the first 20 min and a flow rate of 20 µl/hr for another 20 min. For washing, pump DMEM into the chip at a flow rate of 20 µl/hr for 20 min.
   3. Take photos, via a charge-coupled device (CCD) camera, of certain FOVs within culture areas for analyzing the fluorescent intensities.

5. Calculations of Chemical Concentrations, Shear Stresses, and Electric Fields

1. In both the CSS chip and the CEF chip, calculate the chemical concentrations in the five culture areas. For example, by injecting H₂O₂ of concentrations 0 and 200 µM from the two inlets, concentrations of 0, 25, 100, 175, and 200 µM are generated.
   NOTE: By assuming that all liquids split-flow smoothly and equally around the fork, the relative concentrations in the five culture areas are 0, 1/8, 1/2, 7/8, and 1, respectively.
2. In the CSS chip, calculate the shear stress (τ) within each of the culture areas using ²⁷, where Q is the volume flow rate, η is the fluidic viscosity, h is the height of the channel, and w is the width of the channel.
   NOTE: By setting Q = 0.3 ml/min in each inlet (Q = 0.12 ml/min in each culture area), η = 0.0008 Pa·s for culture medium, h = 1 mm, and w = 1 ~ 4 mm, the shear stress is calculated to range from 0.0048 Pa (4 mm wide region) to 0.0192 Pa (1 mm wide region).
3. In the CEF chip, calculate the EF strength within each of the culture areas using E = I/(σA_eff) (Ohm's law), where I is the electric current flowing across the fluidic channel, σ is the electrical conductivity of the culture medium, and A_eff is the effective cross-sectional area of the channel.
   NOTE: Using σ = 1.38 Ω^-1m^-1 for culture medium and A_eff = 0.22 mm² (width = 1 mm and height = 0.22 mm), the EF strength is calculated to be E (mV/mm) = I (A) × 3.3 × 10⁶.
   1. As shown in Figure 2D, treat the equivalent circuit as five C-section circuits with four (8 + 35 + 8) segments and one (5 + 35 + 5) segment.
      NOTE: By analyzing this parallel circuit according to Kirchhoff's voltage law and Ohm's law, currents flowing across five culture areas, I₁ through I₅ from bottom to top, are calculated to be around 0.49I (area 1), 0.25I (area 2), 0.13I (area 3), 0.08I (area 4), and 0.05I (area 5), respectively, where I is the total direct current (dc). With an applied dc of 0.157 mA, EFs of 254, 130, 67, 41, and 26 mV/mm are generated within the five culture areas.
      NOTE: For a simplified electrical analysis of the microfluidic network, all fluidic segments are considered as resistors with resistance proportional to their lengths.

6. Data Analysis

Note: Data analysis is performed using the ImageJ software.

1. Analyze the Production of ROS.
   1. Run the ImageJ software. Go to "File" → Open to load a fluorescent image to be analyzed.
   2. Go to "Image" → "Type" → "16-bit" to change the image to a gray scale.
   3. Draw a polygon to enclose a cell. Go to "Analyze" → "Measure" to measure the mean fluorescent intensity of the cell.
   4. Repeat 6.1.3 to collect intensities from at least 50 cells of three independent experiments, and calculate the mean intensity with standard error of mean (SEM).
   5. Repeat 6.1.1-6.1.4 for each experimental condition.
2. Analyze Cell Migration.
   1. Run the ImageJ software. Go to "File" → Open to load an image taken at time = 0 to be analyzed.
   2. Draw a polygon to enclose a cell. Go to "Analyze" → "Measure" to measure the center of mass of the cell as (x₁, y₁).
   3. Repeat 6.2.1- 6.2.2 to measure the center of mass of the same cell as (x₂, y₂) from another image take at time = t.
   4. Calculate the migration rate (in µm/hr) of this cell as .
   5. Repeat 6.2.1-6.2.4 to collect migration rates from at least 50 cells of three independent experiments, and calculate the mean migration rate with standard error of mean (SEM).
   6. From 6.2.2 and 6.2.3, calculate the migration directedness of this cell as cosine θ or , where θ is the angle between the vector of the applied EF (from positive to negative) and the vector from the start to the end position of the cell (Figure 2G).
   7. Repeat 6.2.6 to collect migration directedness from at least 50 cells of three independent experiments, and calculate the mean migration directedness with standard error of mean (SEM).
   8. Calculate the mean migration rate with SEM and the mean migration directedness with SEM for each experimental condition.
      NOTE: A directedness of +1 indicates that all cells migrate toward the cathode, and a -1 value indicates that all cells migrate toward the anode. The directedness of a group of randomly moving cells is close to 0.
3. Analyze Cell Alignment.
   1. Run the ImageJ software. Go to "File" → "Open" to load an image to be analyzed.
   2. Treat the cell as an ellipse and draw a line to indicate the long axis of a cell. Go to "Analyze" → "Measure" to measure the angle β between the line and the horizontal EF direction.
   3. Repeat 6.3.2 to collect β from at least 50 cells of three independent experiments, and calculate mean cosine β with standard error of mean (SEM).
   4. Repeat 6.3.1-6.3.3 for each experimental condition.
      NOTE: A cosβ of +1 indicates that all cells align in parallel to the applied EF, and a 0 value indicates that all cells align perpendicularly to the applied EF.

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Results

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, ...

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Discussion

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 ...

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Materials

Name	Company	Catalog Number	Comments
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
ImageJ	National Institutes of Health		Quantifying fluorescent intensities and cell migration