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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Abstract

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

Introduction

Neural precursor cells (NPCs, also known as neural stem and progenitor cells) can be as a promising candidate for neurodegenerative therapeutic strategy1. The undifferentiated NPCs have self-renewal capacity, multi-potency, and proliferative ability2,3. A previous study has reported that the extracellular matrix and molecular mediators regulate differentiation of NPC. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) promote NPC proliferation, thus maintaining the undifferentiated state4.

Previous studies have reported that electrical stimulation can regulate cell physiologic activities such as division5, migration6,7,8, differentiation1,9,10, and cell death11. Electric fields (EFs) play vital roles in the development and regeneration of the central nervous system development12,13,14. From 2009 to 2019, this laboratory has investigated cellular responses to the application of EF in the microfluidic system1,6,7,8,15,16,17. A multichannel, optically transparent, electrotactic (MOE) chip was designed to be suitable for immunofluorescence staining for confocal microscopy. The chip had high optical transparency and good durability and allowed the simultaneous conduct of three independent stimulation experiments and several immunostained conditions in a single study. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This paper describes the development of a microfluidic cell culture system that was used for a long-term cell differentiation study.

Protocol

1. Design and fabrication of the MOE chip

  1. Draw patterns for individual polymethyl methacrylate (PMMA) layers and the double-sided tape using appropriate software (Figure 1A, Table of Materials). Cut both the PMMA sheets and the double-sided tape with a CO2 laser machine scriber (Figure 1B).
    1. Switch on the CO2 laser scriber and connect it to a personal computer. Open the designed pattern file using the software.
    2. Place the PMMA sheets (275 mm x 400 mm) or double-sided tape (210 mm x 297 mm) on the platform of the laser scriber (Figure 2A). Focus the laser onto the surface of the PMMA sheets or the double-sided tape using the auto-focus tool.
    3. Select the laser scriber as the printer, and then "print" the pattern using the laser scriber to start the direct ablation on the PMMA sheet or double-sided tape and obtain individual patterns on the PMMA sheet or tape (Figure 2B).
  2. Remove the protective film from the PMMA sheets, and clean the surface using nitrogen gas.
    NOTE: The drawing of the PMMA pattern and direct machining of the PMMA sheet were performed according to a previous report17.
  3. For bonding together multiple layers of PMMA sheets, stack three pieces of 1 mm PMMA sheets (Layers 1, 2, and 3), and bond them under a pressure of 5 kg/cm2 in a thermal bonder for 30 min at 110 °C to form the flow/electrical stimulation channel assembly (Figure 2C).
    NOTE: Different batches of commercially obtained PMMA sheet have slightly different glass transition temperature (Tg). The optimal bonding temperature needs to be tested at 5 °C increments close to the Tg.
  4. Adhere 12 pieces of adaptors to the individual openings in Layer 1 of the MOE chip assembly with fast-acting cyanoacrylate glue.
    NOTE: The adaptors are made of PMMA by injection molding. The flat surfaces at the bottom are for connecting to the MOE chip. The adaptors bearing 1/4W-28 female screw thread are for connecting white finger-tight plugs, flat bottom connectors, or Luer adaptors. Be careful when using fast-acting cyanoacrylate glue. Avoid splashing into the eyes.
  5. Disinfect the 1 mm PMMA substrates (Layers 1-3), the double-sided tape (Layer 4), and the 3 mm optical grade PMMA (Layer 5) using ultraviolet (UV) irradiation for 30 min before assembling the chip (Figure 1A).
  6. Adhere the 1 mm PMMA substrates (Layers 1-3) on the 3 mm optical grade PMMA (Layer 5) with the double-sided tape (Layer 4) to complete the PMMA assembly (Layers 1-5) (Figure 1A).
  7. Prepare the clean cover glass for the assembly on the chip.
    1. Fill a ten-fold dilution of the detergent in a staining jar (see the Table of Materials), and clean the cover glass in this detergent using an ultrasonic cleaner for 15 min.
    2. Thoroughly rinse the staining jar under running tap water to remove all traces of the detergent.
    3. Continue rinsing with distilled water to remove all traces of tap water, and repeat step 1.7.2 two times.
    4. Dry the cleaned cover glass by blowing it with nitrogen gas.
  8. Disinfect the PMMA assembly (Layers 1-5), the double-sided tape (Layer 6), and the cover glass (Layer 7) using UV irradiation inside a biosafety cabinet for 30 min before assembling the chip (Figure 1A).
  9. Adhere the cleaned cover glass (Layer 7) to the PMMA assembly (Layers 1-5) with the double-sided tape (Layer 6) (Figure 1A).
  10. Incubate the MOE chip in a vacuum chamber overnight; use the MOE chip assembly for subsequent procedures (Figure 3).

2. Coating poly-L-lysine (PLL) on the substrate in the cell culture regions

  1. Prepare the polytetrafluoroethylene tube, flat-bottom connector, cone connector, cone-Luer adaptor, white finger-tight plug (also called stopper), Luer adaptor, Luer lock syringe, and black rubber bung (Figure 4A, Table of Materials). Sterilize all the above components in an autoclave at 121 °C for 30 min.
  2. Seal the openings of the agar bridge adaptors (Figure 1A) with the white finger-tight plugs. Connect the flat-bottom connector to the MOE chip assembly via the medium inlet and outlet adaptors (Figure 4B). Connect the cone-Luer adaptor to the 3-way stopcocks.
  3. Add 2 mL of 0.01% PLL solution using a 3 mL syringe that connects to the 3-way stopcock of the medium inlet (Figure 4B-figure-protocol-5015).
  4. Connect an empty 3 mL syringe to the 3-way stopcock of the medium outlet (Figure 4B-figure-protocol-5234).
  5. Fill the cell culture regions with the PLL solution. Manually pump the coating solution back and forth slowly. Close the two 3-way stopcocks to seal the solution inside the culture regions.
  6. Incubate the MOE chip at 37 °C overnight in an incubator filled with 5% CO2 atmosphere.

3. Preparation of the salt bridge network

  1. Following step 2.6, open the two 3-way stopcocks and flush away the bubbles in the channels by manually pumping the coating solution back and forth in the channel using the two syringes.
  2. Draw 3 mL of complete medium (stem cell maintenance medium consisting of Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (DMEM/F12), 2% B-27 supplement, 20 ng/mL EGF, and 20 ng/mL bFGF) into a 3 mL syringe that connects to the 3-way stopcock of the medium inlet (Figure 4B-figure-protocol-6259 and Figure 4B-figure-protocol-6401).
  3. Add 3 mL of complete medium to replace the coating solution in the cell culture regions. Connect an empty 5 mL syringe to the 3-way stopcock of the medium outlet (Figure 4B-figure-protocol-6709).
  4. Prepare the salt bridge network (Figure 5).
    1. Cut the black rubber bung to produce a gap, and insert the silver (Ag)/silver chloride (AgCl) electrodes through the black rubber bung and into the Luer lock syringe (Figure 4A).
    2. Replace the white fingertight plug with the Luer adaptor, and inject 3% hot agarose to fill the Luer adaptor.
      NOTE: For the preparation of the hot agarose, dissolve 3 g of agarose powder in 100 mL of phosphate-buffered saline (PBS) and sterilize in an autoclave at 121 °C for 30 min.
    3. Connect the Luer lock syringe to the Luer adaptor. Inject 3% hot agarose through the black rubber bung to fill the Luer lock syringe using the syringe with needle. Allow 10 to 20 mins for the agarose to cool down and solidify.
      NOTE: In order to increase the volume capacity of the agarose, the Luer lock syringe is mounted on the Luer adaptor (Figure 4 and Figure 5). Then, the large electrodes are inserted into the Luer lock syringe. The electrode is capable of providing a stable electrical stimulation for the long-term experiment.

4. Preparation of mNPCs

  1. Culture the mNPCs1 in the complete medium in a 25T cell culture flask at 37 °C in an incubator filled with 5% CO2 atmosphere. Subculture the cells every 3-4 days, and perform all experiments with cells that have undergone 3-8 passages from the original source.
  2. Transfer the cell suspension to a 15 mL conical tube, and spin-down the neurospheres at 100 × g for 5 min. Aspirate the supernatant, and wash the neurospheres with 1x Dulbecco's PBS (DPBS). Spin-down the neurospheres at 100 × g for 5 min.
  3. Aspirate the 1x DPBS and then resuspend the neurospheres in the complete medium. Mix thoroughly and gently.
  4. Add 1 mL of the neurosphere suspension using a 1 mL syringe that connects to the 3-way stopcock of the outlet (Figure 4B-figure-protocol-8978).

5. Setup of the microfluidic system for DC pulse stimulation (Figure 6)

  1. Install the cell-seeded MOE chip onto the transparent ITO heater that is fastened on a programmable X-Y-Z motorized stage.
    NOTE: The ITO surface temperature is controlled by a proportional-integral-derivative controller and maintained at 37 °C. A K-type thermocouple is clamped between the chip and the ITO heater to monitor the temperature of the cell culture regions within the chip. The MOE chip is installed on a programmable X-Y-Z motorized stage and is suitable for automatic time-lapse image acquisition at individual channel sections. The fabrication of the ITO heater and the setup of the cell culture heating system have been described previously18,19.
  2. Infuse the mNPCs by manual pumping into the MOE chip via the medium outlet. Incubate the cell-seeded MOE chip on the 37 °C ITO heater for 4 h.
  3. After 4 h, pump the complete medium through the MOE chip via the medium inlet at a flow rate of 20 µL/h using a syringe pump.
    NOTE: The mNPCs are grown and maintained in the chip for an additional 24 h before EF stimulation to allow cell attachment and growth. The waste liquid is collected in an empty 5 mL syringe connected to the 3-way stopcock of the outlet, shown as "waste" in Figure 6A. The MOE microfluidic system configuration is shown in Figure 6. This microfluidic system provides a continuous supply of nutrition to the cells. The complete fresh medium is continuously pumped into the MOE chip to maintain a constant pH value. Therefore, the cells can be cultured outside a CO2 incubator.
  4. Use electrical wires to connect an EF multiplexer to the MOE chip via the Ag/AgCl electrodes on the chip. Connect an EF multiplexer and a function generator to an amplifier to output square-wave DC pulses with a magnitude of 300 mV/mm at a frequency of 100 Hz at 50% duty cycles (50% time-on and 50% time-off) (Figure 6B).
    1. Connect the electrical wires to the EF multiplexer. Connect the electrical wires to the MOE chip via the Ag/AgCl electrodes.
    2. Connect the EF multiplexer to the amplifier using electrical wires. Connect the function generator to the amplifier and the digital oscilloscope.
      NOTE: The EF multiplexer is a circuit that includes the impedance of the culture chamber in the circuit and connects all individual chambers in a parallel electronic network. Each of the three culture chambers is electrically connected in serial to a variable resistor (Vr) and an ammeter (shown as µA in Figure 6A) in the multiplexer. The electric current through each culture chamber is varied by controlling the Vr, and the current is shown on the corresponding ammeter. The electric field strength in each cell culture region was calculated by Ohm's Law, I= σEA, where I is the electric current, σ (set as 1.38 S·m-1 for DMEM/F1220) is the electrical conductivity of the culture medium, E is the electric field, and A is the cross-sectional area of the electrotactic chamber. For the cell culture region dimension shown in Figure 1, the electric current is ~87 mA and ~44 mA for DC and DC pulse at 50% duty cycle, respectively.
  5. Subject the mNPCs to square DC pulses with a magnitude of 300 mV/mm at the frequency of 100 Hz for 48h. Continuously pump the complete medium at a rate of 10 µL/h to supply adequate nutrition to the cells and to maintain a constant pH value in the medium.

6. Immunofluorescence assays of mNPCs after pulsed DC stimulation

NOTE: In this step, all reagent is pumped via the medium inlet using a syringe pump.

  1. After 3, 7, or 14 days in vitro (DIV) culturing after seeding1, wash the cells with 1x PBS at a flow rate of 25 µL/min for 20 min.
  2. Fix the cells with 4% paraformaldehyde (PFA). Pump 4% PFA into the chip at a flow rate of 25 µL/min for 20 min to replace the 1x PBS. To replace the 4% PFA, wash the cells with 1x PBS at a flow rate of 25 µL/min for 20 min.
  3. Pump 0.1% Triton X-100 into the chip at a flow rate of 50 µL/min for 6 min to permeabilize the cells. Reduce the flow rate to 50 µL/h for an additional 30 min to react with the cells. To replace the 0.1% Triton X-100, wash the cells with 1x PBS at a flow rate of 50 µL/min for 6 min.
  4. Block the cells with PBS containing 1% bovine serum albumin (BSA) to reduce nonspecific antibody binding. Pump 1% BSA into the chip at a flow rate of 50 µL/min for 6 min. Reduce the flow rate to 100 µL/h and pump for 1 h.
  5. Pump the antibodies for double immunostaining into the chip at a flow rate of 50 µL/min for 6 min, and incubate the chip for 18 h at 4 °C. Wash the cells with 1x PBS at a flow rate of 50 µL/min for 15 min.
  6. Pump the Alexa Fluor-conjugated secondary antibodies into the chip at a flow rate of 50 µL/min for 6 min. Reduce the flow rate to 50 µL/h, and pump the antibodies for 1 h at room temperature in the dark. Wash the cells with 1x PBS at a flow rate of 50 µL/min for 15 min.
  7. For nuclear staining, pump Hoechst 33342 into the chip at a flow rate of 20 µL/min for 10 min at room temperature in the dark. Wash the cells with 1x PBS at a flow rate of 50 µL/min for 15 min.
  8. After immunostaining, observe the cells using a confocal fluorescence microscope.

7. Image analysis and data processing

  1. Analyze the fluorescent images using software with built-in measurement tools (see the Table of Materials).
  2. Compare the Hoechst-counterstained nuclei (total number of cells) in the control and treatment groups, and calculate the percentage of cells expressing each phenotypic marker.

Results

The detailed configuration of the MOE chip is shown in Figure 1. The microfluidic chip provides a beneficial approach for reducing the experimental setup size, sample volume, and reagent volume. The MOE chip was designed to perform three independent EF stimulation experiments and several immunostaining conditions simultaneously in a single study (Figure 3). In addition, the MOE chip, which has a high optical transparency is suita...

Discussion

During the fabrication of the MOE chip, the adaptors are attached to the Layer 1 of the MOE chip with fast-acting cyanoacrylate glue. The glue is applied to 4 corners of the adaptors, and then pressure is applied evenly over the adaptors. Excess amount of glue must be avoided to ensure complete polymerization of the glue. Moreover, the completed MOE chip assembly is incubated in a vacuum chamber. This step helps to remove the bubbles between the PMMA layer, the double-sided tape, and the cover glass.

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Professor Tang K. Tang, Institute of Biomedical Sciences, Academia Sinica, for his assistance in providing mouse neural stem and progenitor cells (mNPCs). The authors also thank Professor Tang K. Tang and Ms. Ying-Shan Lee, for their valuable discussion on the differentiation of mNPCs.

Materials

NameCompanyCatalog NumberComments
1 mm PMMA substrates (Layers 1-3)BHTK2R20Polymethyl methacrylate (PMMA), http://www.bothharvest.com/zh-tw/product-421076/Optical-PMMA-Non-Coated-BHT-K2Rxx-xx=-thickness-choices.html
15 mL plastic tubeProtech Technology Enterprise Co., LtdCT-15-PL-TWConical bottomed tube with cap, assembled, presterilized
3 mL syringeTERUMODVR-34133 mL oral syringes, without needle
3 mm optical grade PMMA (Layer 5)CHI MEI CorporationACRYPOLY PMMA SheetOptical grade PMMA
3-way stopcockNIPRONCN-3LSterile disposable 3-way stopcock
5 mL syringeTERUMODVR-34105 mL oral syringes, without needle
AdaptorDong Zhong Co., Ltd.CustomizedPMMA adaptor
AgaroseSigma-AldrichA9414Agarose, low gelling temperature
AmplifierA.A. Lab Systems LtdA-304High voltage amplifier
AutoCAD softwareAutodeskEducational VersionDrafting
B-27 supplementGibco12587-010B-27 supplement (50x), minus vitamin A
Basic fibroblast growth factor (bFGF) PeprotechAF-100-18BAlso called recombinant human FGF-basic
Black rubber bungTERUMODVR-3413From 3 mL oral syringes, without needle
Bovine serum albumin (BSA)Sigma-AldrichB4287Blocking reagent 
CentrifugeHSIANGTAICV2060Centrifuge
CO2 laser scriberLaser Tools and Technics Corp. ILS-IIPurchased from http://www.lttcorp.com/index.htm
Cone connectorIDEX Health & ScienceF-120XOne-piece fingertight 10-32 coned, for 1/16" OD natural
Cone-Luer adaptorIDEX Health & ScienceP-659Luer Adapter 10-32 Female to Female Luer, PEEK
Confocal fluorescence microscopeLeica MicrosystemsTCS SP5Leica TCS SP5 user manual, http://www3.unifr.ch/bioimage/wp-content/uploads/2013/10/User-Manual_TCS_SP5_V02_EN.pdf
Digital cameraOLYMPUSE-330Automatic time-lapse image acquisition
Digital oscilloscopeTektronixTDS2024Measure voltage or current signals over time in an electronic circuit or component to display amplitude and frequency.
Double-sided tape3M PET 8018Purchased from http://en.thd.com.tw/
Dulbecco’s modified Eagle’s medium/Ham's nutrient mixture F-12 (DMEM/F12)Gibco12400024DMEM/F-12, powder, HEPES
Dulbecco's phosphate-buffered saline (DPBS)Gibco21600010DPBS, powder, no calcium, no magnesium
EF multiplexerAsiatic Sky Co., Ltd.CustomizedMonitor and control the electric current in individual channels
Epidermal growth factor (EGF)PeprotechAF-100-15Also called recombinant human EGF
Fast-acting cyanoacrylate glue3M 7004TStrength instant adhesive (liquid)
Flat bottom connectorIDEX Health & ScienceP-206Flangeless male nut Delrin, 1/4-28 flat-bottom, for 1/16" OD blue
Function generatorAgilent Technologies33120AHigh-performance 15 MHz synthesized function generator with built-in arbitrary waveform capability
Goat anti-mouse IgG H&L (Alexa Fluor 488)Abcamab150117Goat anti-mouse IgG H&L (Alexa Fluor 488) preadsorbed
Goat anti-rabbit IgG H&L (Alexa Fluor 555)Abcamab150086Goat polyclonal secondary antibody to rabbit IgG - H&L (Alexa Fluor 555), preadsorbed
Hoechst 33342InvitrogenH3570Nuclear staining
ImageJ softwareNational Institutes of Health1.48vAnalyze the fluorescent images 
Indium–tin–oxide (ITO) glassMerck300739For ITO heater
Inverted phase contrast microscopeOLYMPUSCKX41For cell morphology observation
K-type thermocoupleTecpelTPK-02ATemperature thermocouples
Luer adapterIDEX Health & ScienceP618-01Luer adapter female Luer to 1/4-28 male polypropylene
Luer lock syringeTERUMODVR-3413For agar salt bridges
Mouse anti-GFAPeBioscience14-9892Astrocytes marker
Oligodendrocyte  marker  O4  antibodyR&D SystemsMAB1326Oligodendrocytes marker
Paraformaldehyde (PFA)Sigma-AldrichP6148Fixing agent
Phosphate buffered saline (PBS)Basic LifeBL2651Washing solution
Poly-L-Lysine (PLL)SIGMAP4707Coating solution
Precision cover glasses thickness No. 1.5HMARIENFELD107242https://www.marienfeld-superior.com/precision-cover-glasses-thickness-no-1-5h-tol-5-m.html
Programmable X-Y-Z motorised stageTanlian IncCustomizedPurchased from http://www.tanlian.tw/ndex.files/motort.htm
Proportional–integral–derivative (PID) controllerToho ElectronicsTTM-J4-R-ABTemperature controller 
PTFE tubeProfessional Plastics Inc. Taiwan BranchOuter diameter 1/16 InchesWhite translucent PTFE tubing
Rabbit anti-Tuj1Abcamab18207Neuron marker
Syringe pumpNew Era Systems IncNE-1000NE-1000 programmable single syringe pump
TFD4 detergentFRANKLABTFD4Cover glass cleaner
Thermal bonderKuan-MIN Tech Co.CustomizedPurchased from http://kmtco.com.tw/
Triton X-100Sigma-AldrichT8787Permeabilized solution
Ultrasonic cleanerLEOLEO-300SUltrasonic steri-cleaner
Vacuum chamberDENG YNG INSTRUMENTS CO., Ltd.DOV-30Vacuum drying oven
White fingertight plugIDEX Health & ScienceP-3161/4-28 Flat-Bottom, https://www.idex-hs.com/store/fluidics/fluidic-connections/plug-teflonr-pfa-1-4-28-flat-bottom.html

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