Name: Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis (Video) | JoVE Journal
Uploaded: 2013-09-03T14:00:00.000+00:00
Duration: 10 min 38 s
Description: Learn label-free cell sorting using contactless dielectrophoresis (cDEP) for biophysics and microfluidics research. This non-damaging method isolates cells based on intrinsic electrical properties without electrode contact.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

This work supported has been supported in part by the National Science Foundation under Grant No. EFRI 0938047, and by the Virginia Tech Institute for Critical Technology and Applied Science (ICTAS). The authors would like to express appreciation to Dr. Eva Schmelz and Dr. Paul Roberts for their kind gift of MOSE-L cells. The authors acknowledge Angela Anderson for her assistance with cell culture, Caitlan Swaffar for her help with editing this document and preparing experiments, and all Bioelectromechanical Systems lab members.

1. Fabricating a Prototype cDEP Microfluidic Device: Overview
<ol>
	<li>Follow previously reported procedures22 using photoresist and Deep Reactive Ion Etching (DRIE) to etch the desired channel design (Figure 1) into a silicon wafer (Figure 2). Deposit a thin layer of Teflon to improve the release of the microdevice from the wafer.</li>
</ol>
<img alt="Microfluidic chip diagram with sink/source electrodes, dimensions, and fluid flow path analysis." src="/files/ftp_upload/50634/50634fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Schematic of a low frequency continuous sorting device. The sample channel runs left to right and is 500 μm wide with sawtooth constrictions to 100 μm. The two pairs of fluidic electrode channels compose the source and sink electrodes, respectively, and are separated from the sample channel by a 20 μm thick PDMS barrier.
<img alt="Photolithography process diagram: UV exposure, photoresist patterning on silicon, PDMS molding steps." src="/files/ftp_upload/50634/50634fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. The fabrication process of a cDEP device. (a) For 60 sec, UV light selectively reacts with photoresist exposed through a mask pattern of the cDEP device geometry. (b) Photoresist is removed using developer. (c) Deep Reactive Ion Etching (DRIE) is used to etch 50 μm deep features, and 5 min of wet etching by tetramethylammonium hydroxide (TMAH) 25% at 70 °C is used to reduce roughness on the side walls. (d) A Teflon coating is added to improve device release from the wafer. (e) PDMS is poured onto the re-usable wafer stamp after degassing and cured for 45 min at 100 °F. (f) PDMS is removed from the wafer. PDMS and a clean glass slide are exposed to air plasma for 2 min and bonded together.
<ol start="2">
	<li>Wrap the wafer with aluminum foil to prevent spill-over. Mix polydimethylsiloxane (PDMS) in 10:1 ratio of elastomer to curing agent, de-gas for approximately 20 min, and pour onto the wafer. Heat for 45 min at 100 °C to avoid damaging the Teflon coating of the stamp. Allow the device to cool down.</li>
	<li>After cooling, remove aluminum foil, trim PDMS using a surgical blade, and punch access holes at channel inlet/outlet using a blunt puncher suited to the size of tubing. Here, a 1.5 mm blunt puncher is used.</li>
	<li>Clean a glass microscope slide using a rinse of soap and water, ethanol, DI water, and drying with air, respectively. Clean the PDMS device using Scotch Magic tape. Examine under microscope to be sure channels are clean. Expose the slide and PDMS device to air plasma for 2 min. Firmly press channel side of device to glass slide, avoiding formation of air bubbles between the device and the slide (Figure 3).</li>
</ol>
<img alt="Photonic circuit diagram with microstrip line structures for microwave photonics research." fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig3highres.jpg" src="/files/ftp_upload/50634/50634fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. (a) The mask used for photolithography during the silicon wafer stamp fabrication, and (b) the finished PDMS-glass device with the sample and fluid electrode channels filled with green and red food coloring, respectively. See Figure 1 for dimensions. <a href="https://www.jove.com/files/ftp_upload/50634/50634fig3highres.jpg" target="_blank">Click here to view larger figure</a>.
2. Preparing a Cell Suspension in DEP Buffer
<ol>
	<li>Prepare a low conductivity (100 μS/cm) buffer, which will be referred to as "DEP buffer" (8.5% sucrose [w/v], 0.3% glucose [w/v], 0.725% RPMI [v/v]).16</li>
	<li>Suspend cells in DEP buffer at 3 x 106 cells/ml. Here, cancerous late-stage mouse ovarian surface epithelial (MOSE-L) cells are used.</li>
</ol>
Note: Cell viability analysis by the trypan blue dye exclusion method showed a slight decrease in viability (from 95.1-91.6%) of early-stage MOSE (MOSE-E) cells suspended in DEP buffer at room temperature after 5 hr. It is predicted that a similar slight decrease in viability occurs for MOSE-L cells, indicating that cells should not be stored in DEP buffer long-term.
<ol start="3">
	<li>Dye cells using a fluorescent membrane-permeable dye, such as the enzymatically-activated Calcein AM.</li>
	<li>Measure conductivity of the dyed cell suspension. The conductivity should be approximately 100 μS/cm. If the conductivity measurement is too high, spin the sample down, resuspend in DEP buffer at 3 x 106 cells/ml and repeat the conductivity measurement.</li>
</ol>
3. Loading the cDEP Microfluidic Chip
<ol>
	<li>Place the microfluidic chip under vacuum for 30 min.</li>
	<li>Meanwhile, cut a piece of flexible tubing to span the distance from the syringe pump to the microscope stage where the microfluidic chip will be located. Here, a 6 inch length of tubing is required. Fit tubing to needle tip on syringe.</li>
	<li>Estimate the required length for outlet tubing by dividing the target collected sample volume by the cross-sectional area of the tube. Cut the require length of tubing.</li>
	<li>Draw cell suspension into syringe. Check that no bubbles are located in the tubing or syringe.</li>
	<li>Upon removing chip from vacuum, insert the outlet tubing and prime the sample channel quickly to avoid trapped air bubbles in the channels. Gently tap the syringe when priming to avoid rupturing the thin (20 μm) insulating barrier, though it has been observed that the barrier can withstand a constant high throughput near 5 ml/hr without rupturing.</li>
	<li>To prime the electrode channels, quickly place empty pipette tips in one outlet of each fluidic electrode channel. Pipette 200 μl of PBS containing a small amount of rhodamine B and gently tap to introduce the fluid into the opposite end of the electrode channel. Maintain gentle pressure until PBS with rhodamine B visibly progresses up the tip of the empty pipette tip. Repeat for each electrode channel. Rhodamine B is used only to fluorescently visualize the fluidic electrode channels.</li>
	<li>After priming, fill each pipette tip reservoir with PBS with rhodamine B. Avoid bubble formation in the pipette tip reservoirs, and remove any visible bubble by pipetting gently up and down. Detach the outlet tubing and discard appropriately, then insert a fresh outlet tubing reservoir to collect the target volume.</li>
</ol>
4. Characterizing Crossover Frequency of Cells using cDEP Chip
<ol>
	<li>Position the chip on the stage of an inverted microscope equipped with digital camera (Figure 4).</li>
</ol>
<img alt="cDEP chip setup with syringe pump, microscope, electrodes; used for dielectrophoresis experiments." fo:content-width="5in" fo:src="/files/ftp_upload/50634/50634fig4highres.jpg" src="/files/ftp_upload/50634/50634fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. (a) Overall experimental setup for cDEP experiments. The cDEP chip is located on the platform of the inverted microscope. A camera sends images to the computer for recording. The syringe pump maintains a constant inlet flow. The function generator generates a signal which is stepped up by the high voltage amplifier and connected to the cDEP chip by wire electrodes. The oscilloscope monitors the signal sent to the chip. (b) Detail view of the cDEP chip and fluidic and electronic connections. <a href="https://www.jove.com/files/ftp_upload/50634/50634fig4highres.jpg" target="_blank">Click here to view larger figure</a>.
<ol start="2">
	<li>Mount syringe on syringe pump and insert wire electrodes into fluidic electrode channel reservoirs.</li>
	<li>Pump fluid through at 1 ml/hr to ensure good contact between the syringe pump and the syringe. Visually inspect the channel length to ensure unimpeded flow of cells and the absence of bubbles or leaks. Reduce flow rate to 0.005 ml/hr. 
	Note: Throughput as high as 1 ml/hr31,35 has been achieved in devices of other geometries.</li>
	<li>For safety, test the electronics by powering on the function generator, high voltage amplifier, and oscilloscope, and adjusting to desired voltage and frequency. Here these parameters are 200 V and 20 kHz. Upon verification of acceptable performance, power off. Connect electrodes to high voltage amplifier. 
	Note: Observe appropriate electrical safety procedures when operating the electronics at the high voltages used for these experiments.</li>
	<li>Upon achieving steady flow, apply the desired voltage at the desired frequency. In this experiment, voltage is 200 VRMS at frequencies between 5-60 kHz.</li>
	<li>Record video files of the cell response with image processing techniques (step 5) to quantify cell distribution in the channel and to characterize the crossover frequency. To do this, maintain a constant voltage and vary the frequency systematically. At the start and end of the experiment, as well as randomly between experimental runs, record the control cell distribution, the distribution of the cells without applying any electrical field. Estimate the amount of time required for cells to flow from the inlet to the monitored location (here, 5 min). After setting a new frequency, wait this amount of time, then begin recording (here, a 2 min video of cell distribution).</li>
</ol>
5. Image Processing for Characterizing Crossover Frequency
<ol>
	<li>Using a computational script, analyze each video frame by frame to determine the relative y-position (where the z-direction is the vertical depth of the channel, the y-direction is the channel width, and x-direction is the channel length) of each fluorescing cell or particle at a specified x-coordinate downstream from the region of high DEP force in the channel. Create a graph of the relative distribution.</li>
	<li>Compare the centerline of the distribution at each voltage and frequency with the centerline of the control distribution. For a given voltage and varying frequencies, determine the crossover frequency, where the centerline matches the y-position of the control.</li>
</ol>
6. Varying the Experiment to Perform Cell Sorting or Enrichment
<ol>
	<li>Suspend desired mixture in DEP buffer at a total concentration of 3 x 106 particles/ml. Here, this mixture is MOSE-L cells with 4 μm fluorescing beads in DEP buffer. Perform the previously described steps to prepare a cDEP device.</li>
	<li>To sort or enrich cells from a mixture, determine the crossover frequencies of the target cells and the background particles as previously described. Operate the experiment at a frequency between the two crossover frequencies of the target cells and the background particles so that the two populations of particles experience DEP forces in opposite directions. To sort MOSE-L cells and 4 μm beads, operate the microdevice above 11.90±0.63 kHz. the first crossover frequency of MOSE-L cells recently reported by Salmanzadeh et al.33</li>
	<li>To collect the contents of a sorted sample, remove the outlet tubing upon collecting the target volume, by placing a glove finger over the outlet opening and gently tugging on the tubing. Pour the collected target volume into a reservoir for further analysis. Note: Other sample recovery methods could include attaching a permanent reservoir to the chip and removing the sample by simple pipetting.</li>
</ol>

<ol><li>Butler, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aButler%2C+M+%22Cell+culture+and+upstream+processing%22">Cell culture and upstream processing</a>. , Taylor & Francis. (2007).</li>
<li>Pratt, E. D., Huang, C., Hawkins, B. G., Gleghorn, J. P., Kirby, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rare+Cell+Capture+in+Microfluidic+Devices%5BTitle%5D)+AND+%22Chemical+Engineering+Science%22%5BJournal%5D)">Rare Cell Capture in Microfluidic Devices.</a> Chemical Engineering Science. 66, 1508-1522 (2011).</li>
<li>Salmanzadeh, A. D., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+rare+cells+through+their+dielectrophoretic+signature%5BTitle%5D)+AND+%22Journal+of+Membrane+Science+and+Technology%22%5BJournal%5D)">Isolation of rare cells through their dielectrophoretic signature.</a> Journal of Membrane Science and Technology. 3, e112(2013).</li>
<li>Bonner, W. A., Hulett, H. R., Sweet, R. G., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+activated+cell+sorting%5BTitle%5D)+AND+%22The+Review+of+Scientific+Instruments%22%5BJournal%5D)">Fluorescence activated cell sorting.</a> The Review of Scientific Instruments. 43, 404-409 (1972).</li>
<li>Adams, J. D., Kim, U., Soh, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multitarget+magnetic+activated+cell+sorter%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Multitarget magnetic activated cell sorter.</a> Proc. Natl. Acad. Sci. USA. 105, 18165-18170 (2008).</li>
<li>Chabert, M., Viovy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+high-throughput+encapsulation+and+hydrodynamic+self-sorting+of+single+cells%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3191-3196 (2008).</li>
<li>Di Carlo, D., Irimia, D., Tompkins, R. G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+inertial+focusing%2C+ordering%2C+and+separation+of+particles+in+microchannels%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Continuous inertial focusing, ordering, and separation of particles in microchannels.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 18892-18897 (2007).</li>
<li>Di Carlo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inertial+microfluidics%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Inertial microfluidics.</a> Lab Chip. 9, 3038-3046 (2009).</li>
<li>Grover, S. C., Skirtach, A. G., Gauthier, R. C., Grover, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+single-cell+sorting+system+based+on+optical+trapping%5BTitle%5D)+AND+%22J.+Biomed.+Opt%22%5BJournal%5D)">Automated single-cell sorting system based on optical trapping.</a> J. Biomed. Opt. 6, 14-22 (2001).</li>
<li>Lin, S. C. S., Mao, X. L., Huang, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+acoustic+wave+%28SAW%29+acoustophoresis%3A+now+and+beyond%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Surface acoustic wave (SAW) acoustophoresis: now and beyond.</a> Lab Chip. 12, 2766-2770 (2012).</li>
<li>Pethig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+Article-Dielectrophoresis%3A+Status+of+the+theory%2C+technology%2C+and+applications%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Review Article-Dielectrophoresis: Status of the theory, technology, and applications.</a> Biomicrofluidics. 4, (2010).</li>
<li>Gascoyne, P. R. C., Wang, X. B., Huang, Y., Becker, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectrophoretic+separation+of+cancer+cells+from+blood%5BTitle%5D)+AND+%22Ieee+T.+Ind.+Appl%22%5BJournal%5D)">Dielectrophoretic separation of cancer cells from blood.</a> Ieee T. Ind. Appl. 33, 670-678 (1997).</li>
<li>Pohl, H. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPohl%2C+HA+%22Dielectrophoresis+%3A+the+behavior+of+neutral+matter+in+nonuniform+electric+fields%22">Dielectrophoresis : the behavior of neutral matter in nonuniform electric fields</a>. , Cambridge University Press. (1978).</li>
<li>Salmanzadeh, A., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+rare+cells+through+their+dielectrophoretic+signature%5BTitle%5D)+AND+%22Journal+of+Membrane+Science%22%5BJournal%5D)">Isolation of rare cells through their dielectrophoretic signature.</a> Journal of Membrane Science. , Under-review (2012).</li>
<li>Markx, G. H., Talary, M. S., Pethig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+Viable+and+Nonviable+Yeast+Using+Dielectrophoresis%5BTitle%5D)+AND+%22J.+Biotechnol%22%5BJournal%5D)">Separation of Viable and Nonviable Yeast Using Dielectrophoresis.</a> J. Biotechnol. 32 (94), 29-37 (1994).</li>
<li>Flanagan, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unique+dielectric+properties+distinguish+stem+cells+and+their+differentiated+progeny%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Unique dielectric properties distinguish stem cells and their differentiated progeny.</a> Stem Cells. 26, 656-665 (2008).</li>
<li>Labeed, F. H., Coley, H. M., Thomas, H., Hughes, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+multidrug+resistance+reversal+using+dielectrophoresis+and+flow+cytometry%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Assessment of multidrug resistance reversal using dielectrophoresis and flow cytometry.</a> Biophys. J. 85, 2028-2034 (2003).</li>
<li>Hoettges, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectrophoresis-activated+multiwell+plate+for+label-free+high-throughput+drug+assessment%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Dielectrophoresis-activated multiwell plate for label-free high-throughput drug assessment.</a> Anal. Chem. 80, 2063-2068 (2008).</li>
<li>Lapizco-Encinas, B. H., Simmons, B. A., Cummings, E. B., Fintschenko, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulator-based+dielectrophoresis+for+the+selective+concentration+and+separation+of+live+bacteria+in+water%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Insulator-based dielectrophoresis for the selective concentration and separation of live bacteria in water.</a> Electrophoresis. 25, 1695-1704 (2004).</li>
<li>Davalos, R. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Performance+impact+of+dynamic+surface+coatings+on+polymeric+insulator-based+dielectrophoretic+particle+separators%5BTitle%5D)+AND+%22Anal.+Bioanal.+Chem%22%5BJournal%5D)">Performance impact of dynamic surface coatings on polymeric insulator-based dielectrophoretic particle separators.</a> Anal. Bioanal. Chem. 390, 847-855 (2008).</li>
<li>Gallo-Villanueva, R. C., Rodriguez-Lopez, C. E., Diaz-De-La-Garza, R. I., Reyes-Betanzo, C., Lapizco-Encinas, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+manipulation+by+means+of+insulator-based+dielectrophoresis+employing+direct+current+electric+fields%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">DNA manipulation by means of insulator-based dielectrophoresis employing direct current electric fields.</a> Electrophoresis. 30, 4195-4205 (2009).</li>
<li>Shafiee, H., Caldwell, J. L., Sano, M. B., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contactless+dielectrophoresis%3A+a+new+technique+for+cell+manipulation%5BTitle%5D)+AND+%22Biomed+Microdevices%22%5BJournal%5D)">Contactless dielectrophoresis: a new technique for cell manipulation.</a> Biomed Microdevices. 11, 997-1006 (2009).</li>
<li>Park, K., Suk, H. J., Akin, D., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectrophoresis-based+cell+manipulation+using+electrodes+on+a+reusable+printed+circuit+board%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Dielectrophoresis-based cell manipulation using electrodes on a reusable printed circuit board.</a> Lab Chip. 9, 2224-2229 (2009).</li>
<li>Jen, C. P., Maslov, N. A., Shih, H. Y., Lee, Y. C., Hsiao, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Particle+focusing+in+a+contactless+dielectrophoretic+microfluidic+chip+with+insulating+structures%5BTitle%5D)+AND+%22Microsyst+Technol%22%5BJournal%5D)">Particle focusing in a contactless dielectrophoretic microfluidic chip with insulating structures.</a> Microsyst Technol. 18, 1879-1886 (2012).</li>
<li>Hughes, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AC+electrokinetics%3A+applications+for+nanotechnology%5BTitle%5D)+AND+%22Nanotechnology%22%5BJournal%5D)">AC electrokinetics: applications for nanotechnology.</a> Nanotechnology. 11, 124-132 (2000).</li>
<li>Pethig, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectrophoresis%3A+Status+of+the+theory%2C+technology%2C+and+applications%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Dielectrophoresis: Status of the theory, technology, and applications.</a> Biomicrofluidics. 4, 022811(2010).</li>
<li>Pethig, R. Ch. 4. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPethig%2C+R+%22BioMEMS+and+Biomedical+Nanotechnology%22">BioMEMS and Biomedical Nanotechnology</a>. Ferrari, M. auro, Ozkan, M. ihrimah, Heller, M. ichael J. . 4, Springer. US. 103-126 (2007).</li>
<li>Shafiee, H., Sano, M. B., Henslee, E. A., Caldwell, J. L., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+isolation+of+live%2Fdead+cells+using+contactless+dielectrophoresis+%28cDEP%29%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP).</a> Lab Chip. 10, 438-445 (2010).</li>
<li>Salmanzadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectrophoretic+differentiation+of+mouse+ovarian+surface+epithelial+cells%2C+macrophages%2C+and+fibroblasts+using+contactless+dielectrophoresis%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis.</a> Biomicrofluidics. 6, (2012).</li>
<li>Salmanzadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+prostate+tumor+initiating+cells+%28TICs%29+through+their+dielectrophoretic+signature%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Isolation of prostate tumor initiating cells (TICs) through their dielectrophoretic signature.</a> Lab Chip. 12, 182-189 (2012).</li>
<li>Salmanzadeh, A., Sano, M. B., Shafiee, H., Stremler, M., Davalos, R. V. Isolation of Rare Cancer Cells from Blood Cells Using Dielectrophoresis. EMBC 2012., Annual International IEEE Engineering in Medicine and Biology Society, Aug 28-Sep 1, San Diego, , (2012).</li>
<li>Sano, M. B., Caldwell, J. L., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+and+development+of+a+low+frequency+contactless+dielectrophoresis+%28cDEP%29+platform+to+sort+cancer+cells+from+dilute+whole+blood+samples%5BTitle%5D)+AND+%22Biosens.+Bioelectron%22%5BJournal%5D)">Modeling and development of a low frequency contactless dielectrophoresis (cDEP) platform to sort cancer cells from dilute whole blood samples.</a> Biosens. Bioelectron. 30, 13-20 (2011).</li>
<li>Salmanzadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigating+Dielectric+Properties+of+Different+Stages+of+Syngeneic+Murine+Ovarian+Cancer+Cells%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Investigating Dielectric Properties of Different Stages of Syngeneic Murine Ovarian Cancer Cells.</a> Biomicrofluidics. , (2013).</li>
<li>Salmanzadeh, A., Sano, M. B., Shafiee, H., Stremler, M. A., Davalos, R. V. Engineering in Medicine and Biology Society (EMBC), 2012 Annual International Conference of the IEEE, , 590-593 (2012).</li>
<li>Sano, M. B., Salmanzadeh, A., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multilayer+contactless+dielectrophoresis%3A+Theoretical+considerations%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Multilayer contactless dielectrophoresis: Theoretical considerations.</a> Electrophoresis. 33, 1938-1946 (2012).</li>
<li>Millet, L. J., Park, K., Watkins, N. N., Hsia, K. J., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separating+beads+and+cells+in+multi-channel+microfluidic+devices+using+dielectrophoresis+and+laminar%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Separating beads and cells in multi-channel microfluidic devices using dielectrophoresis and laminar.</a> J. Vis. Exp. (48), e2545(2011).</li>
<li>Henslee, E. A., Sano, M. B., Rojas, A. D., Schmelz, E. M., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+concentration+of+human+cancer+cells+using+contactless+dielectrophoresis%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Selective concentration of human cancer cells using contactless dielectrophoresis.</a> Electrophoresis. 32, 2523-2529 (2011).</li>
<li>Salmanzadeh, A., Shafiee, H., Davalos, R. V., Stremler, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+mixing+using+contactless+dielectrophoresis%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Microfluidic mixing using contactless dielectrophoresis.</a> Electrophoresis. 32, 2569-2578 (2011).</li>
</ol>

Dr. Rafael Davalos has a patent pending for contactless dielectrophoresis.

DEP is a powerful technique for determining dielectric properties of particles and directing particle motion towards sorting, isolation, or enrichment applications. Due to the detrimental effect of direct electrode contact with a sample, others have taken approaches similar to the previously presented method to avoid contact. For example, Bashir et al. developed contact-free DEP devices by using a microfluidic PDMS device separated from printed circuit board electrodes by a thin glass coverslip, and this technique is also made available in video format.23,36
Here, we have shown the fabrication of a cDEP chip and fluidic electrodes using a single PDMS substrate, and the experimental protocol for separating ovarian cancer cells from a mixture of cells and fluorescent beads. The presented technique has been successfully used for a variety of more complex and physiologically relevant applications, including sorting live and dead cells,28 tumor initiating cells from prostate cancer cells,30 cancer cells from dilute red blood cells,31,32 and differentiating among stages of breast cancer37 and ovarian cancer.29 cDEP has also been used for mixing particles.38 These applications suggest that by using the simple technique presented, diverse purposes can be accomplished simply by altering the design of the channel geometry.
DEP is useful on the microscale for manipulation of particles.13 For spherical particles, the translational dielectrophoretic force depends upon the size and electrical properties of the particle and its suspending medium, as well as the gradient of the electric field squared:
<img alt="Dielectrophoresis equation; formula for electric field interaction; advanced physics concept." src="/files/ftp_upload/50634/50634eq1.jpg" data-altupdatedat="1787630408000" data-alt="Equation 1"> 
where εm is the permittivity of the suspending medium, r is the radius of the particle, and Rc[k(ω)]is the real part of the Clausius-Mossotti (CM) factor. The CM factor is a measure of the relative polarizability of the particle compared to the suspending medium and determines the direction of the dielectrophoretic force. It is described as
<img alt="Optical excitation formula, \(K(\omega) = \frac{(\varepsilon_p^* - \varepsilon_m^*)}{(\varepsilon_p^* + 2\varepsilon_m^*)}\)." src="/files/ftp_upload/50634/50634eq2.jpg" data-altupdatedat="1787630408000" data-alt="Equation 2"> 
where <img alt="ε&lt;sub&gt;p&lt;/sub&gt;&lt;sup&gt;*&lt;/sup&gt; symbol; used in material science equations and thermodynamic analysis." src="/files/ftp_upload/50634/50634eq3.jpg" data-altupdatedat="1787630408000" data-alt="Equation 3"> and <img alt="Static equilibrium, εᵐ* symbol, formula in physics, methodology illustration for educational use." src="/files/ftp_upload/50634/50634eq4.jpg" data-altupdatedat="1787630408000" data-alt="Equation 4"> are the complex permittivities of the particle and medium, respectively. The complex permittivity, <img alt="Complex permittivity formula ε*=ε+σ/jω for electromagnetic analysis." src="/files/ftp_upload/50634/50634eq5.jpg" data-altupdatedat="1787630408000" data-alt="Equation 5">, depends upon conductivity (σ) and frequency (ω). The CM factor of spherical particles is theoretically bound between -0.5 and 1. If the CM factor is negative, the particles experience nDEP because the medium is more polarizable than the particle, so particles move away from regions of high electric field gradients. If the CM factor is positive, the particles are more polarizable than the medium and they experience pDEP in which they move toward regions of high electric field gradients.
For biological particles that are nonhomogeneous in structure, such as cells, the Clausius-Mossotti factor can be determined from an effective value for the particle permittivity:
<img alt="Static equilibrium equation; dielectric permittivity in layered spheres; formula analysis." src="/files/ftp_upload/50634/50634eq6.jpg" data-altupdatedat="1787630408000" data-alt="Equation 6"> 
where <img alt="Static equilibrium; formula ε*cyt; symbolic representation; educational, research." src="/files/ftp_upload/50634/50634eq7.jpg" data-altupdatedat="1787630408000" data-alt="Equation 7"> and <img alt="Static equilibrium formula, ε*mem, related to membrane potential in spectroscopy analysis." src="/files/ftp_upload/50634/50634eq8.jpg" data-altupdatedat="1787630408000" data-alt="Equation 8"> are the complex permittivity of the effective complex permittivity of the interior of the cell, such as the cytoplasm, and the plasma membrane, respectively; r is the radius of the cell, and d is the thickness of the plasma membrane.26
When DEP occurs with particles suspended in a fluid, the motion of the particle relative to the fluid will generate a drag force on the particle. This drag force must be considered when determining the overall force acting on the particle. For the situations of interest here, viscous forces dominate and particles are assumed spherical, small, and moving with relatively low velocity, so Stokes' drag law provides a good approximation for the drag force:
<img alt="Drag force equation, \( \vec{F}_{\text{Drag}}{=}6\pi\eta r(\vec{u}_p{-}\vec{u}_f) \), physics formula." src="/files/ftp_upload/50634/50634eq9.jpg" data-altupdatedat="1787630408000" data-alt="Equation 9"> 
where η is the viscosity of the fluid, up is the velocity of the particle, and uf is the velocity of the fluid, which may also be moving. Given the dielectrophoretic force, known fluid and particle properties, and a known flow velocity, the balance between the drag force and the dielectrophoretic force can be solved to estimate the particle velocity. The shear rate that the cells experience should be below the threshold at which cell lysing can occur.
Characterization of the electrical properties of particles is necessary to predict and control how they will respond under DEP them. In this work, we specifically utilized low frequency cDEP with MOSE-L cells to demonstrate the protocol for determining first crossover frequency of cells, and then showed continuous sorting of polystyrene beads and MOSE-L cells based on their opposing DEP responses.
Altering the geometry of the cDEP device will change the spatial gradients of the electric field, allowing devices to be designed for high frequency or low frequency operation, and for high selectivity and efficiency of sorting for a specified cell type. Additionally, high throughput devices can be developed by fabricating wider channels,30 channels in parallel,30 or by multilayer fabrication35, in which the electrode channels are vertically stacked above and below a relatively deep sample channel. Thin membranes form the barrier between layers. Preliminary testing with devices fabricated in polymethylmethacrylate (PMMA) and polycarbonate (PC) thin films have demonstrated DEP response of MOSE-L cells. Current efforts are underway to refine multi-layer high-throughput devices and to improve the peripheral system towards an eventual plug-and-play platform. To expand from the basic experimental technique presented, the applications and specifications of the device can be tailored to fit particular demands, such as sorting versus characterization, or adding sample reservoirs and a semi-automated system to the device.

Biological sample enrichment and particle sorting is often necessary for subsequent analysis.1 For instance, isolation of rare cells from body fluids has important applications in cancer detection and individualized medicine. 2,3 The most commonly used enrichment techniques are fluorescent activated cell sorting (FACS)4 and magnetic activated cell sorting (MACS),5 which rely upon expressed surface markers to differentiate cells. Other strategies include hydrodynamic6 or inertial7,8 sorting, optical tweezers,9 acoustophoresis,10 and dielectrophoresis.11,12 Dielectrophoresis is the movement of a polarized particle in the presence of a non-uniform electric field.13 DEP has been used for a wide range of applications,2,14 including sorting cells based on viability,15 characterizing bioelectrical properties of cells,16 and sorting upon induced changes in biophysical properties of cells.17,18 Traditional DEP utilizes planar electrodes patterned within a microfluidic channel to apply a voltage and induce a non-uniform electric field.13 While this is a powerful technique, challenges can arise, such as electrode delamination and electrolysis. Insulator-based dielectrophoresis (iDEP)19 has addressed the challenges of fouling, electrode delamination, and spatial degradation of the electrode field through patterning insulating structures that induce non-uniformities in a DC electric field. iDEP has been used to selectively separate live and dead bacteria cells,19 isolate bacterial spores,20 and manipulate DNA,21 among other applications. Joule heating can be a challenge because it can occur as a result of the high DC voltages often required. To ameliorate these challenges, contact-free DEP microdevices have been developed.22-24
The technique presented here utilizes contactless dielectrophoresis (cDEP), so named for the lack of direct contact between metallic electrodes and the sample channel.22 Unique to this strategy is the replacement of metallic electrodes with fluid electrode channels filled with a highly conductive solution. These fluid electrodes are capacitively coupled across a thin insulating barrier to the sample channel via an AC voltage. Eliminating sample contact with electrodes reduces issues associated with DEP-based methods such as electrolysis and bubble formation, sample contamination, and electrode delamination. As a result, cDEP is especially useful for biological samples because it supports viability of the cells in the sample. Importantly, cDEP can maintain sample sterility. A chip can be prepared in a cell culture hood and the experiment can be conducted without requiring sample contact to metal electrodes or requiring that the sample be open to the environment. A simple reservoir can be fixed to the chip outlet to facilitate sterile sample recovery. Additionally, the fabrication of fluid electrodes from the same biocompatible polymer material (PDMS) as the sample channel reduces the high cost incurred with custom patterning of metal electrodes, the time required for fabrication, and limits the need for specialized cleanroom equipment to the initial patterning of the reusable silicon wafer stamp.
Movement of particles due to DEP depends on the characteristics of the particle and the medium, as well as the spatial gradients of the electric field. A particle- and frequency-dependent factor, called the Clausius-Mossotti (CM) factor, takes a value in the range -0.5 to 1, and determines the direction of the DEP force. The frequency at which the CM factor is exactly zero is called the crossover frequency. This is the point at which no dielectrophoretic force is exerted on a particle and the CM factor changes sign. A single crossover frequency for inert solid microspheres occurs when the CM factor changes from negative to positive. 25 For mammalian cells in low conductivity buffer on the order of 0.01 S/m, a first crossover frequency indicating a transition from nDEP to pDEP exists near 10-100 kHz, and is influenced by the size, shape, cytoskeleton, and membrane properties of the cell.26,27 A second crossover frequency at a shift from the pDEP to nDEP regime is on the order of 10 MHz, and is influenced by the nucleus-cytoplasm ratio, cytoplasm conductivity, and endoplasmic reticulum.27 DEP force can be applied without the presence of fluid flow, but here we utilize a flowing fluid to achieve continuous sorting of suspended particles. The combined influence of the dielectrophoretic force and the Stokes' drag force dictate the translational motion of a particle.
We have developed devices for operation in two frequency ranges. Higher frequency devices (100-600 kHz) have operated using pDEP and achieved batch sorting of cells, such as prostate tumor initiating cells (TICs), murine ovarian surface epithelial (MOSE) cells, MDA-MB-231 breast cancer cells, or live THP-1 cells by selectively trapping cells of interest on insulating posts located in the sample channel.28-31 Lower frequency (5-100 kHz) devices operate continuously, and when operated at a frequency at which one population experiences pDEP while the background population experiences nDEP, can redirect particle trajectories to achieve sorting.32-34 These low frequency devices have been used to sort cancer cells from red blood cells, determine the changes in dielectric properties of a progressive MOSE cell line, and to elucidate the effects of non-toxic sphingolipid treatments on reversing aggressive characteristics of aggressive MOSE cells. Additionally, cDEP microdevices can be designed to operate at increased throughput, currently up to 1 ml/hr.31,35
As described, the flexibility and low cost of the fabrication process enables custom-designed device geometries, which allow the presented experimental procedure to be relevant for a wide range of applications. The long-term goal of cDEP is to realize label-free cell sorting and enrichment on a clinical level, with sample recovery for subsequent culture or processing. The technique presented here is a simple and inexpensive method, from fabrication to experimentation, which increases the accessibility of DEP. We demonstrate the preparation of a cDEP chip and the experimental protocol to achieve characterization and enrichment of ovarian cancer cells from fluorescent microspheres.

<table><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning, Midland, MI,USA</td><td>Sylgard 184</td><td></td></tr><tr><td>Glass slides </td><td>The Microscope Depot</td><td>76079</td><td>2x3 inch-ground edges</td></tr><tr><td>Microbore PTFE Tubing</td><td>Cole-Parmer Instrument Co, Vernon Hills, IL, USA</td><td>EW-06417-31</td><td>Thin walled 20 gauge, 0.032&quot;ID x 0.056&quot;OD, 100 ft/roll</td></tr><tr><td>Luer-slip plastic syringes</td><td>National Scientific company</td><td>S7510-1</td><td></td></tr><tr><td>Needle tip</td><td>Howard Electronic instruments</td><td>JG20-1.0</td><td>20 Gauge 1.0&quot;, ID=0.025&quot; OD=0.036&quot;</td></tr><tr><td>D(+)-Sucrose</td><td>Fisher Scientific, Fair Lawn, NJ</td><td>S3-500</td><td></td></tr><tr><td>D(+)-glucose, reagent ACS, anhydrous</td><td>Acros Organics N.V., Fair Lawn, NJ</td><td>AC410955000</td><td></td></tr><tr><td>RPMI-1640 Medium</td><td>Quality Biological Inc.</td><td>112-025-101</td><td></td></tr><tr><td>Calcein AM, Molecular Probes</td><td>Invitrogen Corp. (life technologies), Carlsbad, CA, USA</td><td>C3100MP</td><td>excitation wavelength 488/emission wavelength 516</td></tr><tr><td>Rhodamine B, O</td><td>Science Lab</td><td>SLR1465-100G</td><td>excitation wavelength 540/emission wavelength 625</td></tr><tr><td>Phosphate buffered saline (10X)</td><td>Gbiosciences, St. Louis, MO</td><td>RC-147</td><td></td></tr><tr><td>Leica, inverted light microscope </td><td>Leica Microsystems, Bannockburn, IL, USA</td><td>Leica DMI 6000B</td><td></td></tr><tr><td>Leica DFC420, color camera</td><td>Leica Microsystems, Bannockburn, IL, USA</td><td>Leica DFC420</td><td></td></tr><tr><td>Function generator </td><td>GW Instek, Taipei, Taiwan</td><td>GFG-3015</td><td></td></tr><tr><td>Wideband power amplifier </td><td>Amp-Line Corp., Oakland Gardens, NY, USA</td><td>AL-50HF-A</td><td></td></tr><tr><td>HFHV Output Transformer</td><td></td><td>AL-T50-V25/300-F100K-600K</td><td></td></tr><tr><td>High voltage amplifier</td><td>Trek</td><td>Model 2205</td><td></td></tr><tr><td>USB Modular Oscilloscope, 100 MHz</td><td>AgilentTechnologies</td><td>U2701A</td><td></td></tr><tr><td>Expanded Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC-001/002 (115/230V)</td><td>air plasma</td></tr><tr><td>Scotch Magic tape</td><td>3M</td><td></td><td>any available width is sufficient</td></tr><tr><td>1.5 mm puncher </td><td>Harris Uni-Core</td><td>Z708836-25EA</td><td></td></tr><tr><td>.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>FluoSpheres Sulfate Microspheres</td><td>Invitrogen</td><td>F8858</td><td>4.0 &amp;mu;m, red fluorescent (excitation wavelength 580/emission wavelength 605)</td></tr><tr><td>AZ 9260 photoresist </td><td>AZ Electronic Materials</td><td></td><td></td></tr><tr><td>AZ 400 K developer</td><td>AZ Electronic Materials</td><td></td><td></td></tr><tr><td>Tetramethylammonium hydroxide (TMAH) 25% </td><td></td><td></td><td>provided by Virginia Tech cleanroom</td></tr><tr><td>Teflon coating</td><td></td><td></td><td>applied using DRIE machine</td></tr><tr><td>Silicon wafer</td><td>University Wafer</td><td>452</td><td>100 mm diameter, 500 &amp;mu;m thickness, one side polished (SSP) </td></tr><tr><td>Deep Reactive Ion Etching (DRIE)</td><td>Alcatrel</td><td>AMS SDE 100</td><td></td></tr></tbody></table>

label-free isolation and enrichment of cells through contactless dielectrophoresis

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

DEP should be observed qualitatively in the sample channel to confirm the device is operational when the electric field is present. One method to test for the presence of DEP is to adjust the frequency to values far from the crossover frequency where strong pDEP and nDEP are predicted (roughly &gt;40 kHz to observe pDEP and &lt;10 kHz to observe nDEP for most mammalian cancer cells in a buffer with conductivity of 100 μS/cm). It should be noted that inert microspheres exhibit pDEP below their crossover frequency and nDEP above their crossover frequency. For the sawtooth feature geometry presented here, pDEP should be seen for cells at the higher test frequency, indicated by the occurrence of pearl chaining and focusing of cells or particles at the sawtooth feature edge of the channel, while at the lower test frequency, nDEP should be apparent as cells are restricted to the region nearer the straight edge of the channel. At these low frequencies, cells may lyse due to electroporation, so the sample may appear to contain fewer cells, and those that are visible may appear enlarged or blurry if using an enzymatically-activated fluorescing dye. Verifying the frequencies at which pDEP and nDEP occur allows the determination of the crossover frequency as described in the protocol. For mammalian cancer cells, such as MOSE-L used here, we observed nDEP at 5 kHz (Figure 5a) and strong pDEP at 60 kHz (Figure 5b). MOSE-L cells had a diameter of 17.7±3.3 μm (n = 268 cells) and the beads have a 4 μm nominal diameter. A full analysis of the dielectric properties of MOSE-L cells compared to MOSE cells in earlier stages of progression can be found in the recent work by Salmanzadeh et al.33
If pDEP and nDEP are not observed at these extremes, various problems could be occurring. The sample conductivity may be too high due to contaminants or cell lysing. Insufficient electric field may be generated due to bubbles trapped in the fluid electrode channels, which prevent conduction of current to the portion of the channels bordering the sample channel. Unsteady flow may be caused by a bubble trapped in the syringe or inlet tubing, bubbles resulting from not keeping the device under vacuum for a sufficient time or not quickly filling the device upon removing from vacuum, leakage due to delamination of poorly bonded PDMS to the glass slide, tears in the barrier membrane due to excessive force exerted when filling the channels, or flow rates at the extremes of the pump operating range.
In addition to determining the crossover frequency of cells, this technique can be used to sort a mixture, illustrated here by a mixture of MOSE-L cells and beads. This example takes advantage of the negative dielectrophoresis (nDEP) exhibited by 4 μm beads at frequencies where MOSE-L cells experience positive dielectrophoresis (pDEP). We operated the device at 200 VRMS and 10 kHz (Figure 6a) and observed that both cells and beads were mixed as they experienced nDEP. At 50 kHz we observed movement of MOSE-L cells to the top of the channel, while beads were restricted to the lower portion of the channel, enabling separation of the mixture into two components (Figure 6b).
<img alt="Fluorescent microscopy of cellular clusters on hydrophobic surface, emphasizing adhesion behavior." src="/files/ftp_upload/50634/50634fig5.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig5highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
Figure 5. The position of MOSE-L cells is manipulated by changing the frequency of the applied electric field. The photos are taken at the final sawtooth feature in the sample channel and the fluidic electrodes are located at the corners on the right side. They are filled with PBS containing rhodamine B, which fluoresces to visualize the channels. (a) MOSE-L cells experience nDEP at 200 VRMS and 5 kHz. (b) MOSE-L cells experience pearl chaining and pDEP at 200 VRMS and 60 kHz. <a href="https://www.jove.com/files/ftp_upload/50634/50634fig5highres.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Fluorescence micrograph of DNA molecules in gel electrophoresis illustrating separation process." src="/files/ftp_upload/50634/50634fig6.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig6highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 6"> 
Figure 6. A mixture of MOSE-L cells (green) and 4 μm beads (red) flow through the final sawtooth feature in the sample channel of a low frequency cDEP device. (a) At 200 V and 10 kHz cells and beads are mixed. Arrows point to faintly appearing cells that likely have died due to the low frequency conditions. (b) At 200 V and 50 kHz cells experience pDEP and move to the feature edge of the channel, while beads experience nDEP and remain confined to the region nearer the straight edge. <a href="https://www.jove.com/files/ftp_upload/50634/50634fig6highres.jpg" target="_blank">Click here to view larger figure</a>.

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

The overall goal of this procedure is to induce translational motion of cells suspended in a pressure driven flow by using dye electrophoresis without contacting electrodes to the sample. This is accomplished by first suspending the cells of interest in a low conductivity buffer. The second step is to load the microfluidic device with the cell suspension and introduce highly conductive fluid into the electrode channels.Next, the prepared device is connected to high voltage electronics and the frequency response of the cell motion is recorded by video fed from a camera attached to an inverted microscope. The final step is to analyze the video images to quantitatively predict electrical properties of the cells. Ultimately, contactless die electrophoresis is used to show that cells with different electrical properties can be sorted in a label-free non-damaging manner, using inexpensive and simple to fabricate microfluidic devices.So this method is useful for applications such as sorting live cells from dead, isolating tumor, initiating cells, separating out cancer cells based on stage and looking at the effects of of drugs on different cells in their dielectric properties. The main advantages of this technique over existing methods such as fluorescence activated cell sorting, is that cells can be sorted and characterized based on their intrinsic biophysical properties without the need for labeling based on biomarkers and also that sample sterility can be maintained by eliminating contact between the electrodes and the sample. Well, we first had this idea when we wanted to develop a technique that avoided several of the issues such as sample contamination, electrolysis expensive fabrication, and that's associated with traditional di electrophoretic devices.For isolating mammalian cells. Follow previously reported procedures using photoresist and deep reactive ion etching to etch the desired channel design into a silicon wafer, depositing a thin layer of Teflon to improve the release of the micro device from the wafer. Wrap the wafer with aluminum foil to prevent spillover.Mix polymethyl alane or PDMS in a 10 to one ratio of elastomer to curing agent DGAs for approximately 20 minutes and pour onto the wafer. Then heat the wafer for 45 minutes at 100 degrees Celsius to avoid damaging the Teflon coating of the stamp after cooling. Remove the aluminum foil.Trim the PDMS using a surgical blade and punch access holes at the channel inlet outlet. Using a blunt puncher suited to the size of the tubing here, a 1.5 millimeter blunt puncher is used. Next, clean a glass microscope slide as directed in the text protocol.Clean the PDMS device using Scotch Magic tape. Examine under a microscope to be sure channels are clean. Then expose the slide and PDMS device to air plasma for two minutes firmly press the channel side of the device to the glass slide, avoiding formation of air bubbles between the device and the slide.Prepare a low conductivity buffer as described in the text protocol, which will be referred to as DEP buffer. Suspend the cells in DEP buffer at 3 million cells per milliliter here cancerous, late stage mouse, ovarian surface epithelial, or moss L cells are used after dying the cells using a fluorescent membrane. Permeable dye measure the conductivity of the dy cell suspension.The conductivity should be approximately 100 microsiemens per centimeter. If the conductivity measurement is too high, spin the sample down. Resus suspend in depth buffer, and repeat the conductivity measurement.The most difficult aspect of this protocol is avoiding bubbles in the electrode channels and the sample channel bubbles in the electrode channels can prevent electro hole connections and bubbles in the sample channel can prevent steady flow. Be sure to perform the demonstrated steps to avoid bubbles and ensure steady flow Before loading the C dip microfluidic chip. Place it under vacuum for 30 minutes.Meanwhile, cut a piece of flexible tubing to span the distance from the syringe pump to the microscope stage where the microfluidic chip will be located here. A six inch length of tubing is required. Fit the tubing to the needle tip on the syringe.Then estimate the required length for outlet tubing by dividing the target collected sample volume by the cross-sectional area of the tube. Cut the required length of tubing. Next, draw the cell suspension into a syringe.Check that no bubbles are located in the tubing or syringe. Upon removing the chip from the vacuum, insert the outlet tubing and prime the sample channel quickly. To avoid trapped air bubbles in the channels, gently tap the syringe when priming.To avoid rupturing the thin insulating barrier to prime the electrode channels quickly place empty pipette tips in one outlet of each fluidic electrode channel pipette 200 microliters of PBS containing a small amount of bromine B.And gently tap to introduce the fluid into the opposite end of the electrode channel. Maintain gentle pressure until PBS with bromine B visibly progresses up the tip of the empty pipette tip, repeat for each electrode. Channel R domine B is used only to fluorescently.Visualize the fluidic electrode channels after priming. Fill each pipette tip reservoir with PBS, with R domine B.Avoid bubble formation in the pipette tip reservoirs and remove any visible bubble by pipetting gently up and down. Detach the outlet tubing and discard appropriately.Then insert a fresh outlet tubing reservoir. Collect the target volume. Position the chip on the stage of an inverted microscope equipped with the digital camera.Mount the syringe on the syringe pump and insert the wire electrodes into the fluidic electrode channel reservoirs pump the fluid through at one milliliter per hour to ensure good contact between the syringe pump and the syringe. Visually inspect the channel length to ensure unimpeded flow of cells and the absence of bubbles or leaks. Then reduce the flow rate to five microliters per hour.For safety. Test the electronics by powering on the function generator, high voltage amplifier and oscilloscope, and adjusting to the desired voltage and frequency. Here these parameters are 200 volts and 20 kilohertz.Upon verification of acceptable performance, shut the power off. Then connect the electrodes to the high voltage amplifier. Upon achieving steady flow, apply the desired voltage at the desired frequency In this experiment, voltage is 200 volts RMS at frequencies between five and 60 kilohertz.Record the video files of the cell response with image processing techniques to quantify cell distribution in the channel and to characterize the crossover frequency. To do this, maintain a constant voltage and vary the frequency systematically at the start and end of the experiment as well as randomly between experimental runs. Record the control cell distribution, which is the distribution of the cells without applying any electrical field.After setting a new frequency wait, the amount of time required for cells to flow from the inlet to the monitored location and then begin recording, proceed to perform the image processing for characterizing crossover frequency as described in the text protocol, suspend the desired mixture in DEP buffer at a total concentration of 3 million particles per milliliter. Here this mixture is moss L cells with four micrometer fluorescent beads in DEP buffer perform the previously described steps. To prepare a CEP device, operate the experiment at a frequency between the two determined crossover frequencies of the target cells and the background particles so that the two populations of particles experience DEP forces in opposite directions.To collect the contents of assorted sample, remove the outlet tubing upon collection of the target volume by placing a glove finger over the outlet opening and gently tugging on the tubing. Pour the collected target volume into a reservoir for further analysis. For mammalian cancer cells, such as the most L cells used here, negative DEP was observed at five kilohertz and strong positive DEP.At 60 kilohertz MOS L cells have a diameter of 17.7 plus or minus 3.3 micrometers, and the beads have a four micrometer nominal diameter. In addition to determining the crossover frequency of cells, this technique can be used to sort a mixture illustrated here by a mixture of MO'S L cells and beads. This example takes advantage of the negative dielectrophoresis exhibited by four micrometer beads at frequencies where MO'S L cells experience positive dielectrophoresis.When the device was operated at 200 volts RMS and 10 kilohertz, both cells shown here in green and beads shown here in red were mixed as they experienced negative DEP at 50 kilohertz. Movement of MOS L cells to the top of the channel was observed while beads were restricted to the lower portion of the channel, enabling separation of the mixture into two components. So following this procedure, other techniques can be conducted downstream, such as cell culturing and immunofluorescence imaging or biochemical assays to to answer additional questions such as how the dielectric properties correlate to their physical characteristics of the cells.So after its development, this technique really paved the way for biologists to study how metabolite induced changes in the cytoskeleton of the cell can be correlated to their dielectric properties using a mouse ovarian cancer model. After watching this video, you should have a good understanding of how to enrich and sort cells using contactless electrophoresis.

label-free-isolation-enrichment-cells-through-contactless

Research

JoVE Journal

Medicine

Cell Capture Using a Microfluidic Device

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.

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

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

A Method of Targeted Cell Isolation via Glass Surface Functionalization

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation and Activation of Murine Lymphocytes

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer's patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells.

Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis

Here, we present light-induced dielectrophoresis as a label-free approach for characterizing heterogeneous cell lines, specifically human mesenchymal stem cells (hMSCs). This paper describes a protocol for using and optimizing a microfluidic device with a photoconductive layer to characterize the electrical behavior of hMSCs without altering their native state.

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Learn label-free cell sorting using contactless dielectrophoresis (cDEP) for biophysics and microfluidics research. This non-damaging method isolates cells based on intrinsic electrical properties without electrode contact.

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Explore More Videos