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.

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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.

<table><tbody><tr><td>Acetone</td><td>J.T. Baker</td><td>5356-05</td><td>Purity (GC)&amp;nbsp; &amp;ge; 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></tr><tr><td>Analog Low-Pass Filter</td><td>ThorLabs</td><td>EF504</td><td>&amp;le;240 kHz Passband, Coaxial BNC Feedthrough&amp;nbsp;(https://www.thorlabs.com/thorproduct.cfm?partnumber=EF504#ad-image-0)</td></tr><tr><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><tr><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&amp;amp;utm_source=google&amp;amp;utm_&lt;br/&gt; medium=cpc&amp;amp;utm_campaign=&lt;br/&gt; Shopping_Product_Cable%20Assemblies_NEW&amp;amp;utm_term=&lt;br/&gt; &amp;amp;utm_content=Coaxial%20Cables%20%28RF%29&amp;amp;gclid=Cj0KCQjwlK-WBhDjARIsAO2sErQqnVJ&lt;br/&gt; pj5OXVObuTI8ZUf1ZeIn7zvzGnx&lt;br/&gt; mCWdePrG6SdEJMF3X6ubUaAs&lt;br/&gt; w-EALw_wcB</td></tr><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&amp;amp;products_&lt;br/&gt; 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&amp;nbsp;</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&amp;nbsp;</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Spectrum Chemical</td><td>I1056-4LTPL</td><td>Purity (GC)&amp;nbsp; &amp;ge;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)&amp;nbsp; &amp;ge;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&amp;ndash;1.10 mm&amp;nbsp;</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 &amp;amp; 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&quot; ID x 0.056&quot;&amp;nbsp;(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&amp;amp;gclid=&lt;br/&gt; Cj0KCQjwlK-WBhDjARIsAO&lt;br/&gt; 2sErRwzrrgjU0NjFkDkne1xm&lt;br/&gt; vT7ekS3tdzvAgiMDwPoxocgH&lt;br/&gt; 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&amp;nbsp;</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 ...

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

Research

JoVE Journal

Bioengineering

2.6K Views.  University of California, Berkeley and University of California, San Francisco. 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.

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

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Developmental Biology

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Biology

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Neuroscience

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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

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A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro