This research was supported by grants from NIBIB 1R01EB024989-01 and NCI 1R01CA190843-01. A. L. and R. R. were supported by an H2H8 Association Graduate Research Fellowship. K. L. C. was supported by a National Science Foundation Graduate Research Fellowship and a Siebel Scholar Fellowship.

1. Design device geometry
<ol>
	<li>Choose the width of the sizing and recovery segments so that it is wider than the diameter of the largest cells to be measured but also maintains a sufficient signal-to-noise ratio (SNR). See Supplementary Table 2 for examples of different sizing and recovery segment widths for various cell lines.</li>
	<li>Choose the contraction segment width to apply a 30%-40% strain to the average size of the cells that are to undergo mechanophenotyping. Strain is...

<ol>
	<li>Pegoraro, A. F., Janmey, P., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+cytoskeleton+and+cells.">Mechanical properties of the cytoskeleton and cells.</a> Cold Spring Harbor Perspectives in Biology. 9 (11), 022038 (2017).</li><li>Kim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+cellular+mechanical+phenotypes+with+mechano-node-pore+sensing.">Characterizing cellular mechanical phenotypes with mechano-node-pore sensing.</a> Microsystems &amp; Nanoengineering. 4, 17091 (2018).</li><li>Mierke, C. 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IFMBE Proceedings. 23, 2122-2125 (2009).</li><li>Rother, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy-based+microrheology+reveals+significant+differences+in+the+viscoelastic+response+between+malign+and+benign+cell+lines.">Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines.</a> Open Biology. 4 (5), 140046 (2014).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM+indentation+study+of+breast+cancer+cells.">AFM indentation study of breast cancer cells.</a> Biochemical and Biophysical Research Communications. 374 (4), 609-613 (2008).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elasticity+measurement+of+breast+cancer+cells+by+atomic+force+microscopy.+Proc.+SPIE+9230.">Elasticity measurement of breast cancer cells by atomic force microscopy. Proc. SPIE 9230.</a> Twelfth International Conference on Photonics and Imaging in Biology and Medicine. (PIBM 2014). 92300, (2014).</li><li>Alcaraz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrheology+of+human+lung+epithelial+cells+measured+by+atomic+force+microscopy.">Microrheology of human lung epithelial cells measured by atomic force microscopy.</a> Biophysical Journal. 84 (3), 2071-2079 (2003).</li><li>Li, M., Dang, D., Liu, L., Xi, N., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+in+characterizing+cell+mechanics+for+biomedical+applications:+A+review.">Atomic force microscopy in characterizing cell mechanics for biomedical applications: A review.</a> IEEE Transactions on Nanobioscience. 16 (6), 523-540 (2017).</li><li>Urbanska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+microfluidic+methods+for+high-throughput+cell+deformability+measurements.">A comparison of microfluidic methods for high-throughput cell deformability measurements.</a> Nature Methods. 17, 587-593 (2020).</li><li>Hill, R. T., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Patterning.">Surface Patterning.</a> Biomaterials Science: An Introduction to Materials: Third Edition. , 276-301 (2013).</li><li>Wang, Z., Volinsky, A. A., Gallant, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+effect+on+polydimethylsiloxane+elastic+modulus+measured+by+custom-built+compression+instrument.">Crosslinking effect on polydimethylsiloxane elastic modulus measured by custom-built compression instrument.</a> Journal of Applied Polymer Science. 131 (22), 41050 (2014).</li><li>Gibson, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hierarchical+structure+and+mechanics+of+plant+materials.">The hierarchical structure and mechanics of plant materials.</a> Journal of the Royal Society Interface. 9 (76), 2749-2766 (2012).</li><li>Stephens, A. D., Banigan, E. J., Adam, S. A., Goldman, R. D., Marko, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+and+lamin+a+determine+two+different+mechanical+response+regimes+of+the+cell+nucleus.">Chromatin and lamin a determine two different mechanical response regimes of the cell nucleus.</a> Molecular Biology of the Cell. 28 (14), 1984-1996 (2017).</li><li>Rosenbluth, M. J., Lam, W. A., Fletcher, D. 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L. L. S holds US patent No. 11,383,241: "Mechano-node-pore sensing", J. Kim, S. Han, and L. L. Sohn, issued July 12, 2022.

Measuring the mechanical properties of single cells using this mechanophenotyping technique consists of three stages: device fabrication, data acquisition, and data analysis. Within each stage, there are notable aspects that may significantly impact the experimental results. During device fabrication, consistent channel geometries and device-to-device uniformity are essential for accurate and repeatable results. Specifically, the sidewalls of each device should be relatively smooth (Figure 4Ai</stron...

Single cells are dynamic, viscoelastic materials1. A multitude of internal and external processes, (e.g., onset of mitosis or remodeling of the extracellular matrix [ECM]), influence their structure and composition2,3,4, often resulting in distinct biophysical properties that complement their current state. In particular, mechanical properties have been shown to be important biomarkers of cellular development, physiology, and pathology, yielding valuable quantitative information that can supplement canonical molecular and genetic approaches5,6,7. For example, Li et al. recently described the mechanical differences between drug-resistant and drug-responsive acute promyelocytic leukemia cells, while also using RNA-seq to uncover differentially-expressed cytoskeleton-associated genes8. By understanding the complex interplay between single-cell mechanics and cellular function, mechanophenotyping has broader applications in transforming basic science and clinical diagnostics9.
The most widely adopted tool for measuring single-cell mechanics is atomic force microscopy (AFM). While AFM enables a high-resolution, localized measurement of cellular mechanical properties, it remains limited to a throughput of &#60;0.01 cells/s10. Alternatively, optical stretchers, which use two divergent laser beams to trap and deform suspended single cells11, are limited to marginally higher throughputs of &#60;1 cell/s12. Recent advances in microfluidic technologies have enabled a new generation of devices for rapid, single-cell, mechanical assessment12,13. These techniques employ narrow constriction channels14,15, shear flow16, or hydrodynamic stretching17 to deform cells quickly at throughputs of 10-1,000 cells/s18. While the measurement rate of these approaches is considerably faster than conventional techniques, they often trade high-throughput capabilities for limited mechanical readouts (Supplementary&#160;Table 1).&#160;All the aforementioned rapid microfluidic methods focus on basic, single-parameter metrics, such as transit time or deformability ratios, that only reflect a cell&#39;s elastic properties. However, given the intrinsic viscoelastic nature of single cells, a robust and thorough mechanical characterization of cells requires consideration of not only elastic components but also viscous responses.
Mechano-node-pore sensing (mechano-NPS)2,8 (Figure 1A) is a microfluidic platform that addresses existing limitations with single-cell mechanophenotyping. This method enables the measurement of multiple biophysical parameters simultaneously, including cell diameter, relative deformability, and recovery time from deformation, with a moderate throughput of 1-10 cells/s. This technique is based on node-pore sensing (NPS)19,20,21,22,23,24, which involves using a four-point probe measurement to measure the modulated current pulse produced by a cell transiting a microfluidic channel that has been segmented by wider regions, referred to as &#34;nodes&#34;. The modulated current pulse is a result of the cell partially blocking the flow of current in the segments (i.e., &#34;pores&#34;) and nodes, with more current blocked in the former than in the latter. In mechano-NPS, one segment, the &#34;contraction channel&#34;, is narrower than a cell diameter; consequently, a cell must deform to transit the entire channel (Figure 1B). Cell diameter can be determined by the magnitude of the subpulse produced when the cell transits the node-pores prior to the contraction channel (Figures 1B,C). Here, |&#916;Inp|, the current drop when the cell is in the pore,&#160;is proportional to the volume ratio of the cell to the pore, Vcell/Vpore2,8,19. Cell stiffness can be determined by &#916;Tc, the duration of the dramatically larger subpulse produced when the cell transits the contraction channel (Figures 1B,C). A stiffer cell will take longer to transit the channel than a softer one2,8. Finally, cell &#34;recovery&#34;, the cell&#39;s ability to return to its original size and shape post deformation, can be determined by the series of subpulses produced as the cell transits the node-pores after the contraction channel (Figures 1B,C). The recovery time, &#916;Tr, is the time it takes for the current subpulses to return to the magnitude of the previous subpulses, prior to the cell being squeezed. Overall, the modulated current pulses produced as a cell transits the microfluidic channel are recorded and analyzed to extract the relevant single-cell mechanical parameters (Figure 1D)2,8.
The reproducibility and ease of use of this electronics-based microfluidic platform have been previously demonstrated25. Additionally, the platform presents a low barrier to entry for single-cell mechanophenotyping. Standard soft lithography is employed to fabricate microfluidic devices. The measurement hardware consists of inexpensive components, including a simple printed circuit board (PCB), power supply, preamplifier, data acquisition board (DAQ), and computer. Finally, user-friendly code is available for data acquisition and analysis, enabling straightforward implementation. This mechanophenotyping technique can distinguish populations of non-malignant and malignant breast and lung epithelial cell lines, discriminate between sublineages in primary human mammary epithelial cells, and characterize the effects of cytoskeletal perturbations and other pharmacological agents2,8. Overall, this platform is an effective approach for the mechanophenotyping of single cells.

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			<td>Acetone</td>
			<td>J.T. Baker</td>
			<td>5356-05</td>
			<td>Purity (GC)&#160; &#8805; 99.5% (https://us.vwr.com/store/product/6057739/acetone-99-5-vlsi-j-t-baker)</td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
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			<td>Analog Low-Pass Filter</td>
			<td>ThorLabs</td>
			<td>EF504</td>
			<td>&#8804;240 kHz Passband, Coaxial BNC Feedthrough&#160;(https://www.thorlabs.com/thorproduct.cfm?partnumber=EF504#ad-image-0)</td>
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			<td>Biopsy Punch</td>
			<td>Integra Miltex</td>
			<td>33-31AA-P/25</td>
			<td>1mm, Disposable, with Plunger (https://mms.mckesson.com/product/573313/Miltex-33-31AA-P25)</td>
		</tr>
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			<td>Blade</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC Cable</td>
			<td>Pomona Electronics</td>
			<td>2249-C-12</td>
			<td>https://www.digikey.com/en/products/detail/pomona-electronics/2249-C-12/603323?utm_adgroup=Coaxial%20Cables%20%28RF%29&#38;utm_source=google&#38;utm_ 
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			&#38;utm_content=Coaxial%20Cables%20%28RF%29&#38;gclid=Cj0KCQjwlK-WBhDjARIsAO2sErQqnVJ 
			pj5OXVObuTI8ZUf1ZeIn7zvzGnx 
			mCWdePrG6SdEJMF3X6ubUaAs 
			w-EALw_wcB</td>
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		<tr>
			<td>Cleanroom Polyester Swab</td>
			<td>Thermo Fisher Scientific</td>
			<td>18383</td>
			<td>https://www.fishersci.com/shop/products/texwipe-cleantip-alpha-polyester-series-swabs-6/18383</td>
		</tr>
		<tr>
			<td>Current Preamplifier</td>
			<td>DL Instruments</td>
			<td>1211</td>
			<td>https://www.brltest.com/index.php?main_page=product_info&#38;products_ 
			id=1419</td>
		</tr>
		<tr>
			<td>Custom PCB (w/ components)</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>see Supplemental files 4 and 5</td>
		</tr>
		<tr>
			<td>DAQ Terminal Block</td>
			<td>National Instruments</td>
			<td>BNC-2120</td>
			<td>https://www.ni.com/en-in/support/model.bnc-2120.html</td>
		</tr>
		<tr>
			<td>DAQ to BNC-2110 cable&#160;</td>
			<td>National Instruments</td>
			<td>SHC68-68-EPM</td>
			<td>https://www.ni.com/en-in/support/model.shc68-68-epm.html</td>
		</tr>
		<tr>
			<td>Data Acquisition Board (DAQ)</td>
			<td>National Instruments</td>
			<td>PCI-6251</td>
			<td>https://www.ni.com/docs/en-US/bundle/pci-6251-feature/page/overview.html</td>
		</tr>
		<tr>
			<td>Dessicator</td>
			<td>Thermo Fisher Scientific</td>
			<td>5311-0250</td>
			<td>https://www.thermofisher.com/order/catalog/product/5311-0250</td>
		</tr>
		<tr>
			<td>Female BNC To Banana Plug Adapter</td>
			<td>Pomona Electronics</td>
			<td>72909</td>
			<td>https://www.digikey.com/en/products/detail/pomona-electronics/72909/1196318</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td>https://us.vwr.com/store/product/18706419/avantor-seradigm-select-grade-usda-approved-origin-fetal-bovine-serum-fbs</td>
		</tr>
		<tr>
			<td>Glass Cutter</td>
			<td>Chemglass</td>
			<td>CG-1179-21</td>
			<td>https://chemglass.com/plate-glass-cutters-diamond-tips</td>
		</tr>
		<tr>
			<td>Gold Etchant TFA</td>
			<td>Transene</td>
			<td>NC0977944</td>
			<td>https://www.fishersci.com/shop/products/NC0977944/NC0977944</td>
		</tr>
		<tr>
			<td>Hot Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>SP131825&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Spectrum Chemical</td>
			<td>I1056-4LTPL</td>
			<td>Purity (GC)&#160; &#8805;99.5% (https://www.spectrumchemical.com/isopropyl-alcohol-99-percent-fcc-i1056)</td>
		</tr>
		<tr>
			<td>Metal Hardware Enclosure</td>
			<td>Hammond Manufacturing</td>
			<td>EJ12126</td>
			<td>https://www.digikey.com/en/products/detail/hammond-manufacturing/EJ12126/2423415</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Purity (GC)&#160; &#8805;99.8% (https://www.sigmaaldrich.com/IN/en/substance/methanol320467561)</td>
		</tr>
		<tr>
			<td>MF-321 Developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/mf-321/</td>
		</tr>
		<tr>
			<td>MICROPOSIT S1813 Positive Photoresist</td>
			<td>DuPont</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/microposit-s1800-g2-series-photoresists/</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>https://www.thermofisher.com/order/catalog/product/10010049?SID=srch-hj-10010049</td>
		</tr>
		<tr>
			<td>Photomask</td>
			<td>Fineline Imaging</td>
			<td>n/a</td>
			<td>Photomask are custom ordered from our CAD designs (https://www.fineline-imaging.com/)</td>
		</tr>
		<tr>
			<td>Plain Glass Microscope Slide</td>
			<td>Fisher Scientific</td>
			<td>12-553-5B</td>
			<td>Material: Soda Lime, L75 x W50 mm, Thickness: 0.90&#8211;1.10 mm&#160;</td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td>https://harrickplasma.com/plasma-cleaners/expanded-plasma-cleaner/</td>
		</tr>
		<tr>
			<td>Plastic Petri Dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB0875712</td>
			<td>100 mm (https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-raised-ridge-100-x-15mm/FB0875712)</td>
		</tr>
		<tr>
			<td>Pressure Controller</td>
			<td>Fluigent</td>
			<td>MFCS-EZ</td>
			<td>https://www.fluigent.com/research/instruments/pressure-flow-controllers/mfcs-series/</td>
		</tr>
		<tr>
			<td>Pressure Controller Software</td>
			<td>Fluigent</td>
			<td>MAESFLO</td>
			<td></td>
		</tr>
		<tr>
			<td>Programming &#38; Computation Software</td>
			<td>MATLAB</td>
			<td>R2021b</td>
			<td>for data acquisition and analysis (https://www.mathworks.com/products/matlab.html)</td>
		</tr>
		<tr>
			<td>PTFE Tubing</td>
			<td>Cole Parmer</td>
			<td>06417-31</td>
			<td>0.032&#34; ID x 0.056&#34;&#160;(https://www.coleparmer.com/i/masterflex-transfer-tubing-microbore-ptfe-0-032-id-x-0-056-od-100-ft-roll/0641731)</td>
		</tr>
		<tr>
			<td>Scepter 2.0 Handheld Automatic Cell Counter</td>
			<td>Millapore Sigma</td>
			<td>PHCC20060</td>
			<td>https://www.sigmaaldrich.com/IN/en/product/mm/phcc20060</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>Wafer World</td>
			<td>2885</td>
			<td>76.2 mm, Single Side Polished (https://www.waferworld.com/product/2885)</td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025 Negative Photoresist</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/su-8-2000/</td>
		</tr>
		<tr>
			<td>SU8 Developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td>https://kayakuam.com/products/su-8-developer/</td>
		</tr>
		<tr>
			<td>Sygard 184 Polydimethlysiloxane</td>
			<td>Dow Chemical</td>
			<td>4019862</td>
			<td>https://www.ellsworth.com/products/by-market/consumer-products/encapsulants/silicone/dow-sylgard-184-silicone-encapsulant-clear-0.5-kg-kit/</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Scotch</td>
			<td>810-341296</td>
			<td>https://www.staples.com/Scotch-Magic-Tape-810-3-4-x-36-yds-1-Core/product_130567?cid=PS:GS:SBD:PLA:OS&#38;gclid= 
			Cj0KCQjwlK-WBhDjARIsAO 
			2sErRwzrrgjU0NjFkDkne1xm 
			vT7ekS3tdzvAgiMDwPoxocgH 
			VTQZi7vJgaAvQZEALw_wcB</td>
		</tr>
		<tr>
			<td>Titanium, Platinum, Gold</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Output Power Supply</td>
			<td>Keysight</td>
			<td>E36311A</td>
			<td>https://www.newark.com/keysight-technologies/e36311a/dc-power-supply-3o-p-6v-5a-prog/dp/15AC9653</td>
		</tr>
		<tr>
			<td>UV Mask Aligner</td>
			<td>Karl Suss America</td>
			<td>MJB3 Mask Aligner&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechano-node-pore sensing: a rapid, label-free platform for multi-parameter single-cell viscoelastic measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

The mechanophenotyping platform presented here is a simple and versatile approach for measuring the biophysical properties of single cells with moderate throughput. Cells are flowed through the microfluidic channel (Figure 1A) using constant pressure-driven flow. As the cells transit, the length of the microfluidic channel and the current pulses produced are recorded using the data acquisition hardware. The acquired signal (Figure 1B,C) is then ...

Watch this Scientific Journal Video about Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements at JoVE.com

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

We present an electronics-based microfluidic platform for mechanical phenotyping of single cells. Specifically, we measure single cell elastic and viscous properties at a throughput of up to 10 cells per second. Our method requires minimal sample preparation, utilizes a straightforward electronic measurement, replacing expensive optical hardware and complex image analysis, and is non-destructive, meaning our approach is compatible with downstream analyses.Mechano-NPS has been applied to many cell types, including primary samples, and has measured the effect of subcellular components on single cell viscoelasticity. It could be used for understanding cell behavior, determining disease progression, or monitoring drug response. To begin, remove the plasma treated components from the plasma chamber.Pipet a two to one solution of methanol and deionized water on the glass substrate with pre-fabricated electrodes. Then place the PDMS mold with the feature side down on top of the glass substrate. Place the device under the stereoscope to adjust alignment.Bake the aligned device to complete device fabrication. Prepare the pressure source, PCB, benchtop hardware, and data acquisition software. Next, align the clamps header pins with the holes on the PCB and align the clamps spring loaded pins with the electrode contact pads on the microfluidic device.Then insert the clamps header pins into the PCB holes, making sure the spring loaded pins stay aligned with the electrode contact pads. Set up and connect the electronic hardware. Next, set the values to initialize the data acquisition session and set the sample rate for acquisition.Culture and prepared the cells according to the appropriate cell culture protocol of the cell line of choice. Then suspend the cells in a prepared solution of 2%FBS and 1X PBS at a concentration between 100, 000 and 500, 000 cells per milliliter. Keep the cells on ice for the duration of the experiments.To load cells into the microfluidic device, cut 30 centimeters of polytetrafluoroethylene tubing with a razor blade. Use a syringe to drop the cell sample into the end of the tubing and connect the same end into the device inlet. Finally, connect the opposite end of the tubing to the microfluidic pressure controller.To run the experiment, set the desired constant driving pressure on the pressure controller software and allow the sample to fill the device. If bubbles form in the microfluidic channels, use dead-end filling. Plug the device outlet and apply a low pressure to the inlet to force air out through the gas permeable PDMS.Then set the desired voltage by rotating the voltage knob on the power supply and enable the voltage by pressing the on button. Turn on the current preamplifier and set the sensitivity as low as possible. Alternatively, set the gain as high as possible without overloading the preamplifier or exceeding the maximum analog input voltage of the DAQ.Press the green Run button in the MATLAB ribbon menu to begin the data acquisition script NPS. m, and start sampling and saving the data. To end the experiment, press the Stop button in the lower left corner of the live plot window to end the data acquisition script.Then disable the power supply output by pressing the On button. Finally, set the pressure source to zero pressure in the pressure controller software. For data analysis, the raw signal should have a sufficient signal-to-noise ratio to filter out the noise and extract the significant components.Critically, the current signal rise from each node should be robust enough such that subpulses can be easily identified from the difference signal. Malignant MCF-7 cells have a greater wCDI distribution than non-malignant MCF-10A cells, indicating that the malignant MCF-7 cells are softer than their nonmalignant MCF-10A counterparts. MCF-10A and MCF-7 cells treated with latrunculin show an increase in wCDI.A distinct wCDI distribution differentiating the two primary cell types indicates that LEP cells are softer than MEP cells. Nonmalignant MCF-10As and untreated MCF-10A and MCF-7s have a greater proportion of cells that recover instantly, indicating lower viscosity than their malignant or latrunculin treated counterpart. If the live current readout appears abnormal, stop your experiment and inspect the microfluidic channel.Ensure that there are no air bubbles and no clogs such that the cells are flowing freely from the inlet to the outlet. Because our method doesn't harm cells, downstream analyses like RNA-seq or immunofluorescence can be performed. This could help uncover some underlying reasons why cells have distinct mechanical phenotypes.

mechano-node-pore-sensing-rapid-label-free-platform-for-multi

Research

JoVE Journal

Bioengineering

2.5K Görüntüleme.  University of California, Berkeley and University of California, San Francisco. Tek hücrelerin mekanik fenotiplemesi için elektronik tabanlı bir mikroakışkan platform sunuyoruz. Spesifik olarak, tek hücreli elastik ve viskoz özellikleri, saniyede 10 hücreye kadar bir verimde ölçüyoruz. Yöntemimiz minimum numune hazırlama gerektirir, pahalı optik donanımın ve karmaşık görüntü analizinin yerini alan basit bir elektronik ölçüm kullanır ve tahribatsızdır, yani yaklaşımımız aşağı akış analizleriyle uyumludur.Mekano-NPS, birincil örn...