Name: separating beads and cells in multi-channel microfluidic devices using dielectrophoresis and laminar flow
Uploaded: 2011-02-04T18:15:00
Duration: 9 min 45 s
Description: Watch this Scientific Journal Video about Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow at JoVE.com

We express appreciation to Woo-Jin Chang for providing the trifurcating channels for DEP actuation and to Mitchell Collens for his assistance in equipment setup. The work has been supported by US NSF through Grant OISE-0951647 and by Taiwan NSC through Grant 99-2911-1-002-007.

A general diagram of the equipment setup is shown in Figure 1A. The PCB-sample assembly is further detailed in a cross-sectional schematic in Figure 1B.
1. Preparing PCB Electrodes:
<ol>
<li>Design PCB electrodes to the desired geometry to generate a non-uniform electrical field. Customized PCB electrode chips can be ordered through commercial fabrication facilities (Figure 1C).</li>
<li>Prepare pre-fabricated PCB electrodes by soldering 16-gauge wire to the end of each printed metal electrode (Figure 1D).
<ol>
<li>Place the wire onto the end of the electrode. Hold the wire in place on the metal area of the PCB with the hot soldering iron to heat the wire.</li>
<li>Feed a small amount of solder into the heated wire to fill the wire with solder.</li>
<li>After the wire is filled with solder, remove the soldering iron, holding the wire in place while the solder cools. </li></ol>
</li>
<li>Repeat the soldering process for each electrical connection on the PCB (Figure 1D).</li>
</ol>
2. Prepare Microfluidic Channels:
<ol>
<li>Polydimethylsiloxane (PDMS)-based microfluidic channels are prepared using the PDMS elastomer, Sylgard 184 from Dow Corning. A master mold which defines the channels is usually created through standard microfabrication processes using a silicon wafer and SU-8 photoresist.</li>
<li>Mix base compound to curing agent at a 10:1 ratio for 5 minutes.</li>
<li>Pour the liquid PDMS onto the prefabricated SU-8 master mold and remove air bubbles by exposing liquid PDMS to vacuum for a few minutes. Repeat vacuum process if needed to completely remove all bubbles.</li>
<li>Cure the PDMS at 70 &deg;C for 2 hours.</li>
<li>Remove PDMS slab with microfluidic channels from the wafer with a razor blade, careful not to break wafer.</li>
<li>Punch holes for introducing fluids and cells into the microfluidic device. (Note: Syringe pumping, gravity or surface-tension based flow can all be used with DEP.)</li>
<li>Inspect the microfluidic device to ensure it is free of dust and debris. Cleaning the PDMS can be easily achieved using 3M Scotch Magic Tape.</li>
<li>Plasma bond the PDMS microfluidic channels to a clean no.0 thickness (80-130 &mu;m) coverslip. Heat the coverslip-microfluidic assembly at 100 &deg;C for a minimum of 15 min.</li>
</ol>
3. Prepare Low-conductivity Media:
<ol>
<li>Low-conductivity media is prepared by mixing 8.5% sucrose + 0.3% glucose (w/v) in deionized (DI) water.1 
Note: We have previously demonstrated that cells can be cultured for days in conventional cell culture media after being exposed to low conductivity for short periods (approximately 30 min).11</li>
</ol>
4. Assemble Microfluidic Channels Onto PCB Electrodes:
<ol>
<li>Place a small amount (approximately 10 &mu;L) of mineral oil onto the PCB to ensure tight contact between the PCB and the coverslip. 
Note: An optional step for decreasing electrode visualization is to coat the PCB surface with a very thin layer of black permanent marker or paint.</li>
<li>Place the coverslip-microfluidic channel assembly onto the oiled PCB, with coverslip forming contact with the oil (coverslip down). Gently press the coverslip-microfluidic assembly down to ensure a good contact and to minimize air bubbles that can detract from cell and bead visualization. An example of the completed device is shown in Figure 1D.</li>
</ol>
5. Sort and Concentrate Beads and Cells using DEP:
<ol>
<li>Fill the microfluidic channels with DI water or low-conductivity media; the plasma bonding process facilitates the easy loading of aqueous solutions into the microfluidic channels by temporarily making the normally hydrophobic PDMS surface hydrophilic.</li>
<li>Introduce cells and/or polystyrene beads into the channel reservoir (Figure 1C). Here we use human colon adenocarcinoma (HT-29) cells.</li>
<li>Connect the output of a function generator to the input of an AC power amplifier, then connect the output of the amplifier to the electrode wires. Cover all electrical wires and surfaces of the setup with electrical tape to protect users from the potential exposure to shock. A schematic diagram of the equipment setup is shown in Figure 1A.</li>
<li>Set the function generator to produce a sine wave output of 1.0-1.5 MHz. The amplitude of the output should be adjusted for the RF power amplifier to produce an output of 80-100V to the PCB. The RF power amplifier in this work requires an input voltage around 220-330 mV. To separate cells and beads, the laminar flow rate must be compliant with the DEP force to move the cells and beads within the main channel (width w = 100 &mu;m, height h = 27 &mu;m) into the target channels (width w = 100 &mu;m, height h = 27 &mu;m). 
Caution: consult with PCB manufacturer to determine the maximum operating voltages to avoid issues such as PCB overheating or melting. Check the equipment manufacturers' specifications for the function generator and power amplifier manufacturers to determine safe operating settings.</li>
<li>Initiate DEP to sort cells and beads. Cells experience a positive-DEP whereas polystyrene beads experience a negative-DEP in a non-uniform electric field within the specified frequencies. This enables DEP-force-activated bioactuation and separation of cells and other particles within microfluidic devices using affordable and reusable PCBs as electrodes.</li>
</ol>
6. Representative Results:
When DEP is effective at actuating cells or particles, a robust alignment within non-uniform electrical fields is observed for static baths or slow fluid velocities. Under conditions of higher fluid velocities, the cell or particle behavior depends on the relative orientation of the axial flow to the electrical field and the flow velocity. In addition, the cell and particle behaviors are also dependent upon the electrical field strength and the degree of non-uniformity within the electrical field. Typical behaviors of cells or particles include 'pearl chains,' cycling, stalling, or turning. 
Conditions that compromise or abolish DEP include the presence of salts or other ionic molecules in the fluid, weak electric field strength, excessive flow velocities, coverslips that are too thick, or conductive solutions between the coverslip and PCB electrodes (e.g. a cracked coverslip can induce the mixing of water and mineral oil).
Here, when using no.0 thickness coverslips (80-130 &mu;m) and an electrode spacing of (231 &mu;m), the gradient of the square of the electric field intensity applied to the HT-29 cells and beads is estimated to be between 2-8 to 6-8 V2/&mu;m3.1
The DEP force can be estimated by measuring the velocity of the particle, manipulated by DEP in static fluid (Figure 2A-B). Due to the small inertia of the particle and highly viscous environment of microfluidic channel, the hydrodynamic drag force will be equal to, but in opposite direction with the DEP force. The average bead velocity in the low-conductivity media is 34.5 &mu;m/s with DEP voltage of 93 Vpp and 1.5 MHz. The hydrodynamic drag force can be calculated with following equation.1
Fdrag = 6&pi;&eta;Rv
The average viscosity &eta; of the low-conductivity media at 20 &deg;C is 1.27 mPa&#8226;s and the bead radius R is 7.5 &mu;m. The hydrodynamic drag force for the polystyrene microbead is estimated to be 6.19 pN. To demonstrate the ability to actuate beads within microfluidic channels (Figure 2C-F), we initiated the flow of the low-conductivity media by employing surface tension mediated flow. The average bead velocity in the single input channel was 1540 &mu;m/sec, whereas the average velocity within the individual channels was reduced to 565 &mu;m/sec (Figure 2C). By initiating DEP, the beads were retained within the central channel rather than being released into the side channel. Thus, under these conditions, the DEP forces were sufficient to overcome the drag force of the fluid into the two side channels.
The same principle was also used to divert the beads from the central cannel to a single side channel by simply changing the orientation of the channels and the bead flow to that of the DEP electrodes (Figure 2E-F). By angling the metal electrode on the side of the single inlet channel, when DEP was initiated, the beads were guided to the side of the channel as the beads approached the trifurcation point. There, the DEP forces were sufficient to pull the beads into one of the side channels where the laminar flow continued to propel them down the channel (Figure 2F).
Because cells and polystyrene beads have distinct differences in their ability to be polarized and actuated in non-uniform electrical fields, we use DEP to demonstrate the ability to perform the on-chip separation of cells and beads, simultaneously. We used the same microfluidic channel structure and PCB electrodes as configured in Figure 2E-F, but introduced a suspension of HT-29 cells and beads in low-conductivity media into the single inlet channel (Figure 3). When DEP is introduced, and under these conditions, the HT-29 cells were retained in the two left output channels whereas the beads were retained in the individual output channel on the right. Here the cells show a characteristic 'pearl-chaining' behavior as they are collected in the left output channel. Occasionally a bead and cell become attached together in which they are collected together, often in the cell output channels. From this experimental data, we determine the sorting rate to be 713 particles (cells and beads) per minute, or the equivalent of 14,260 cells and beads in 20 minutes. The number of cells and beads retrieved is relevant and useful for molecular biology, imaging, biochemical and lab-on-a-chip applications.
 <img src="/files/ftp_upload/2545/2545fig1.jpg" alt="Figure 1" /> 
Figure 1. PCB-based DEP of cells and particles in microfluidic channels. (A) The equipment setup for DEP-based actuation starts with a function generator to define the frequency and amplitude of the electrical signal, then a power amplifier to boost the signal strength of the electrical field generated on the PCB. (B) The microfluidic device assembly for sample actuation consists of PDMS microfluidic channels irreversibly bound to a no. 0 thickness coverslip (80-130 &mu;m) through oxygen plasma, non-conductive media to bathe the cells and/or beads. (C) The PCB electrodes used here consist of two regions where the electrodes are interdigitated to generate a strong non-uniform electric field. (D) The completed device: a PCB with a trifurcated microfluidic device on a single coverslip, inset shows the whole device with electrode wires. (C-D) For scale, PCB measurements are 8.4 cm (l), 2.1 cm (w), with 5 mm-wide electrodes. 
 <img src="/files/ftp_upload/2545/2545fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative images of cells and beads in channels before and during DEP actuation. (A) 15 &mu;m fluorescent polystyrene beads in a low-conductivity media solution within a PDMS microfluidic channel, without laminar flow (time lapsed, 1.23 sec). (B) Upon DEP initiation, the beads migrate toward the PCB electrode patterns (black stripes) rather than between electrodes (time lapsed, 8.18 sec). (C) Beads in the same channels under laminar flow are divided between the three separate channels (time lapsed, 5.3 sec); when DEP is initiated (D), the beads are actuated to flow only in the central channel (time lapsed, 4.3 sec). (E) By changing the orientation of the channels to the PCB electrodes, beads can be differentially directed into the side channels (F) using DEP rather than the central channel as shown in (D). (E-F) Time lapsed is 8.23 sec and 5.16 sec, respectively. All scale bars = 100 &mu;m.
 <img src="/files/ftp_upload/2545/2545fig3.jpg" alt="Figure 3" /> 
Figure 3. Representative images of cells and beads in channels before and during DEP actuation. (A-B) Prior to initiating DEP, a mixed solution of human colon adenocarcinoma (HT-29) cells and beads flow from one channel to 3 separate output channels. The open arrow identifies the direction of laminar flow and channels are outlined with dashed lines for orientation and clarification. (C-D) By inducing DEP, beads and cells are selectively actuated into separate channels as identified. HT-29 cells exit the central and left channel whereas beads exit the right channel. Characteristic pearl chaining of beads and cells is observed during DEP actuation. (A, C) Differential Interference Contrast imaging of cells and beads in microchannels renders the beads easily visible. Glow scale intensity images (B, D) of the same DIC images (A, C) improve the visualization of cells in channels. The reflective metal electrodes ((A, C) light stripe and (B, D) yellow and green) provide a prominent landmark for aligning microchannels to the electrodes for effective DEP-based actuation. Scale bars = 100 &mu;m.

<ol>
	<li>Park, K., Suk, H. J., Akin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoresis-based+cell+manipulation+using+electrodes+on+a+reusable+printed+circuit+board.">Dielectrophoresis-based cell manipulation using electrodes on a reusable printed circuit board.</a> Lab Chip. 9, 2224-2224 (2009).</li><li>Nilsson, J., Evander, M., Hammarstrom, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+et+al.,+Review+of+cell+and+particle+trapping+in+microfluidic+systems."> et al., Review of cell and particle trapping in microfluidic systems.</a> Anal. Chim. Acta. 649, 141-141 (2009).</li><li>Fu, A. Y., Chou, H. P., Spence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+microfabricated+cell+sorter.">An integrated microfabricated cell sorter.</a> Anal. Chem. 74, 2451-2451 (2002).</li><li>Rhee, S. W., Taylor, A. M., Cribbs, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+force-assisted+cell+positioning+inside+microfluidic+devices.">External force-assisted cell positioning inside microfluidic devices.</a> Biomed. Microdevices. 9, 15-15 (2007).</li><li>Zhang, H., Liu, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+tweezers+for+single+cells.">Optical tweezers for single cells.</a> J. R. Soc. Interface. 5, 671-671 (2008).</li><li>Hunt, T. P., Westervelt, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoresis+tweezers+for+single+cell+manipulation.">Dielectrophoresis tweezers for single cell manipulation.</a> Biomed. Microdevices. 8, 227-227 (2006).</li><li>Vahey, M. D., Voldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+equilibrium+method+for+continuous-flow+cell+sorting+using+dielectrophoresis.">An equilibrium method for continuous-flow cell sorting using dielectrophoresis.</a> Anal. Chem. 80, 3135-3135 (2008).</li><li>Burgarella, S., Bianchessi, M., Merlo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modular+Platform+for+Cell+Characterization,+Handling+and+Sorting+by+Dielectrophoresis.">A Modular Platform for Cell Characterization, Handling and Sorting by Dielectrophoresis.</a> Cytometry A. 77A, 189-18 (2010).</li><li>Shafiee, H., Sano, M. B., Henslee, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+of+live/dead+cells+using+contactless+dielectrophoresis+(cDEP).">Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP).</a> Lab Chip. 10, 438-438 (2010).</li><li>Vahey, M. D., Voldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Cell+and+Particle+Characterization+Using+Isodielectric+Separation.">High-Throughput Cell and Particle Characterization Using Isodielectric Separation.</a> Anal. Chem. 81, 2446-2446 (2009).</li><li>Park, K., Jang, J., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+cantilever+arrays'+for+characterization+of+mass+of+single+live+cells+in+fluids.">Living cantilever arrays' for characterization of mass of single live cells in fluids.</a> Lab Chip. 8, 1034-1034 (2008).</li><li>Gomez-Sjoberg, R., Morisette, D. T., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+microbiology-on-a-chip:+Microfluidic+bioprocessor+for+rapid+detection+of+bacterial+metabolism.">Impedance microbiology-on-a-chip: Microfluidic bioprocessor for rapid detection of bacterial metabolism.</a> J. Microelectromech. Syst. 14, 829-829 (2005).</li><li>Li, H. B., Zheng, Y. N., Akin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+modeling+of+a+microfluidic+dielectrophoresis+filter+for+biological+species.">Characterization and modeling of a microfluidic dielectrophoresis filter for biological species.</a> J. Microelectromech. Syst. 14, 103-103 (2005).</li></ol>

No conflicts of interest declared.

Cell manipulation in microfluidic devices is desirable for the sorting or selective placement of single cells or for population studies.2 Laminar flow is used in conjunction with valves and pumps to manipulate cells within microfluidic devices. However, these methods alone are challenging and require detailed fabrication processes and skills.3 Centrifugation can simplify the demands for cell placement but simultaneous imaging is a challenge; furthermore, the channel architecture for centrifugation must be considering carefully when designing the desired manipulations and considering effects of centripetal forces.4 Laser tweezers can be used for cell placement but the method is expensive and not amenable for high-throughput cell sorting.5 Yet, DEP has been demonstrated as an effective system of "electrical tweezers" for effective cell placement, characterization and manipulation.6,7
Specifically, DEP has been used to selectively capture and sort cells for on-chip processing of living and dead cells,7-10 and for collecting cells on resonant sensors for the measurement of cell mass.11 We have previously demonstrated that by increasing the DEP force on bacteria or beads above the fluidic drag force, the on-chip concentration and trapping of polystyrene beads and Listeria monocytogenes V7 can be accomplished.12 Mixed populations of E. coli and L. monocytogenes bacteria can also be directed and released through DEP pulses. Furthermore, larger particles can be differentially captured and concentrated on the electrodes based on the large particle size, whereas smaller particles are not capture but are removed with the fluidic flow.13 When DEP forces do not overcome the fluidic dragging forces on the particles, the bead or cell is not captured, but rather moved within the fluidic stream. As shown in Figure 2C, beads can be focused into the central region of the fluid stream sufficient to retain the beads in the central channel. This could be due to the combined effect of particle-to-particle influences within the electrical field, fluid velocities greater than DEP dragging forces, the combination of these, or some other undefined effect.
Newer advances in contactless DEP allow for maximizing cell capture and manipulation with the minimum required field, thus protecting cell types, to the greatest extent, during DEP manipulation.1,9 Contactless DEP offers promise to the microfluidics community for sorting, collecting and positioning cells within microfluidic devices. We anticipate that with the increased demand for, and implementation of, DEP for manipulation within microfluidic devices, further discoveries and innovations will expand the understanding and influence of DEP forces.
PCBs can be manufactured through high-volume processes at an affordable price with a rapid fabrication turnaround time, making them good platforms for commercialization. In addition, PCBs are easy to use and accessible for scientists in a range of disciplines for on-chip cell manipulations and sorting.
When designing the layout of the PCB electrodes, consideration should be given to the desired particle/cell trajectory. Key factors for consideration include particle size and type (bead and/or cell), cell type, microchannel size, flow velocity, electrode spacing (which determines the electrical field strength), coverslip thickness, and the fluid conductivity. These factors influence the force required and available to manipulate the particle or cell, and ultimately the separation efficiency. Our protocol presented here demonstrates an effective setup for initiating DEP for microsphere and cell separation. For potential applications, users should match the architecture of the microfluidic channel design with the desired flow paths for cells and beads, as well as the electrode patterns to optimize efficiency for each application. Electrode spacing and coverslip thickness can be used according to the previously reported guidelines when designing the microfluidic channel layout.1
The conductivity and permittivity of the cell/particle and the surrounding media must be different enough to facilitate positive or negative DEP, while keeping the cells intact. The polarity of DEP for a spherical particle can be determined from the real part of a complex value of the following Clausius-Mosotti factor at the frequency &#969; of DEP voltage.
 <img src="/files/ftp_upload/2545/2545eq1.jpg" alt="Equation 1" /> 
Equation 1
In this equation &#949;, is permittivity, &#963; is conductivity, and &#949;* is complex permittivity. The subscripts p and m denote the particle and media, respectively. When a cell has a higher permittivity than the media, or the real part of the Clausius-Mosotti factor becomes positive, the particle becomes more polarized than the surrounding media. Due to a non-uniform electric field, the particle's polarization becomes non-uniform and this creates a positive DEP (+DEP) force that draws the cell toward the region with higher electric field intensity. If a cell has a lower permittivity than the surrounding media, or the real part of the Clausius-Mosotti factor becomes negative, it will undergo negative DEP (-DEP), and the cell is forced toward the minimal field region. If the cell and media have approximately the same complex permittivity, no force can be generated to manipulate the cell. For this reason, pure water is a preferred media for the DEP particle manipulation. However, to avoid the osmotic stress on the cell from pure water, low conductivity media was formulated to keep the conductivity unchanged, but to increase osmolarity to decrease osmotic stress to the cells. Conventional cell culture media or physiological buffers, such as DMEM or PBS, have high conductivity, which is not suitable for the DEP manipulation.
We also have previously demonstrated that cells can be captured on sensors with DEP using low-conductivity media. After a brief period for cell attachment, the low conductivity media can be replaced for the required cell culture media to support cell growth for days.11
From our experience, fluorescent beads are very bright with respect to the live cell fluorescence, thus it can be a challenge to match the bead intensity to the intensity of a living and fluorescing cell. To improve the visualization of both the cells and the beads, we used DIC microscopy on an upright microscope for imaging both. To display the cells and beads, we presented the data in an intensity glow-scale image, which retains the data in a wide color spectrum for easier viewing. Thus, when designing the study of interest, the imaging parameters and resources must be considered.
In summary, we demonstrate the ability to selectively actuate beads and cells into separate channels using DEP. With the increasing utility of microfluidic channels for cell biology, biochemistry and bioengineering applications, DEP is a desirable option for cell collection, placement and sorting. The PCB electrodes fabrication is inexpensive and convenient, the electrodes are easy to use, and the rapid fabrication times are ideal for the implementation of DEP.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sylgard 184 kit (PDMS)</td>
<td>&nbsp;</td>
<td>Elsworth Adhesives</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>Dow Corning Sylgard</td>
</tr>
<tr>
<td>16 awg hook-up wire</td>
<td>&nbsp;</td>
<td>Belden</td>
<td>8521</td>
<td>(Type MW) Mil-W-76C-PVC</td>
</tr>
<tr>
<td>Gold seal Cover Glass </td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>260320</td>
<td>No. 0 thick, 24 x 60 mm</td>
</tr>
<tr>
<td>Miltex Biopsy Punch</td>
<td>&nbsp;</td>
<td>VWR Scientific</td>
<td>95039-104</td>
<td>Miltex, assorted sizes</td>
</tr>
<tr>
<td>Custom PCB electrodes</td>
<td>&nbsp;</td>
<td>Bay Area Circuits </td>
<td>Custom parts</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>O121-1 </td>
<td>&nbsp;</td>
<tr>
<td>Deionized water</td>
<td>&nbsp;</td>
<td>House DI water supply</td>
<td>None</td>
<td>Use filtered DI water</td>
</tr>
<tr>
<td>D-Glucose Anhydrous Granular AR</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals </td>
<td>4912-06</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose, Crystal</td>
<td>&nbsp;</td>
<td> Mallinckrodt Chemicals</td>
<td>8360-06 </td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent polymer microspheres </td>
<td>&nbsp;</td>
<td> Bangs Laboratories</td>
<td> FS07F/9277</td>
<td>15&mu;m Dragon green microspheres</td>
</tr>
<tr>
<td>HT-29 cell line</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-38D</td>
<td>Human colon adenocarcinoma</td>
</tr>
<tr>
<td>Typsin 0.05% EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>25300054</td>
<td>&nbsp;</td>
<tr>
<td>Eclipse E600FN upright microscope </td>
<td>&nbsp;</td>
<td> Nikon</td>
<td>Eclipse E600FN </td>
<td>&nbsp;</td>
<tr>
<td>Phantom V310 High-speed imaging camera </td>
<td>&nbsp;</td>
<td>Phantom</td>
<td> V310</td>
<td>&nbsp;</td>
<tr>
<td>BX51 upright research microscope </td>
<td>&nbsp;</td>
<td>Olympus </td>
<td>BX51</td>
<td>&nbsp;</td>
<tr>
<td>SpotFlex high resolution color camera </td>
<td>&nbsp;</td>
<td>Diagnostic Instruments </td>
<td>FX1520</td>
<td>&nbsp;</td>
<tr>
<td>Function generator</td>
<td>&nbsp;</td>
<td>Agilent </td>
<td>3325A</td>
<td>&nbsp;</td>
<tr>
<td>RF Power amplifier</td>
<td>&nbsp;</td>
<td>EIN</td>
<td>2100L</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

separating beads and cells in multi-channel microfluidic devices using dielectrophoresis and laminar flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Watch this Scientific Journal Video about Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow at JoVE.com

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

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Research

JoVE Journal

Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

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-1&#945;) 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 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 ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...