Name: pattern generation for micropattern traction microscopy
Uploaded: 2022-02-17T14:00:00.000+00:00
Duration: 9 min 26 s
Description: Watch this Scientific Journal Video about Pattern Generation for Micropattern Traction Microscopy at JoVE.com

The authors would like to thank Dr. Paul Barbone from the Boston University Department of Mechanical Engineering for helpful discussions and assistance with data analysis. This study was supported by NSF grant CMMI-1910401.

The authors have no conflicts of interest to disclose.

An improved method of indirectly patterning PAA hydrogels is described in this paper. This approach builds on methods that have been used previousely20,35,36,37,38,39,40,41,42. The primary change is that PDMS stamps are no...

Most cell phenotypes exert traction forces on their environment. These traction forces are generated by a cell&#39;s contractile cytoskeleton, which is a network of actin and myosin, and other filamentous biopolymers and crosslinking proteins1,2,3,4. Forces generated within the cell can be transmitted to the extracellular environment or adjacent cells, primarily via transmembrane proteins such as integrins and cadherins, respectively5,6. How a cell spreads or contracts-and the magnitudes of the traction forces associated with those movements-is the result of an intimate conversation with its environment, which largely depends on the type and quantity of protein present in the extracellular matrix (ECM)7,8 and the stiffness of the ECM. Indeed, traction force microscopy has become an invaluable tool for understanding cell responsiveness to local stimuli such as substrate stiffness, imposed mechanical stresses and strains, or contact with other cells. This information is directly relevant to the understanding of diseases such as cancer and asthma9,10,11,12.
A system that can be used to measure force-induced deformation of a substrate of known material properties is required to calculate traction forces. These changes must be tracked over time, requiring both imaging and image processing techniques. One of the first methods used to determine cellular traction forces was the observation and analysis of the contraction of collagen hydrogels seeded with cells, though this method was only semiquantitative13. Another, more refined method was to measure the traction forces exerted by single cells by determining the forces resulting from the deformation of a thin sheet of silicone14. Later on, more quantitative measurement techniques were developed, and these methods also allowed for the use of soft hydrogels such as polyacrylamide (PAA)12,15,16. When using these soft materials, traction forces could be determined from the force-induced displacement of randomly displaced beads embedded in the hydrogel and the mechanical properties of the gel16,17. Another advancement came with the development of micropost arrays made of soft polydimethylsiloxane (PDMS) so that their deflection could be measured and converted to force using the beam theory18.
Finally, methods for micropatterning soft hydrogels were developed as these approaches allow control of the contact areas for cell adhesion. By measuring the deformation of the micropattern within a cell&#39;s contact area, traction forces could easily be calculated because a force-free reference image is not required19. This method has been widely adopted as it allows for the indirect patterning of a regular array of micron-sized, discrete fluorescent protein adhesion points onto PAA gels for the measurement of cellular traction forces20. To calculate these forces, an image-processing algorithm, which can track the movements of each micropatterned dot without requiring user input, has been developed21.
While this method is simple for creating entire grids of dot patterns, it is more complicated when patterns of isolated patches (or islands) of dots are desired. Micropatterned islands are useful when control of shape, and to some extent of size, of clusters of cells is needed. To create these islands, the aforementioned method of microcontact printing necessitates two distinct steps: i) using one PDMS stamp to create a high-fidelity pattern of dots on a coverslip, and then ii) using a second different PDMS stamp to remove most of those dots, leaving behind isolated islands of dots21. The difficulty in creating islands with this original method is compounded by the fact that making consistent grid patterns in the first step of the process is challenging on its own. Microprinting stamps are composed of an array of circular microposts, the diameter of which corresponds to the desired dot size. These stamps are then coated with an even layer of protein and then stamped with a precise amount of pressure onto treated coverslips to create the desired pattern. On the one hand, applying too much pressure to the stamp can result in uneven protein transfer and poor pattern fidelity due to pillar buckling or sagging between pillars, leading to contact with the glass. On the other hand, applying too little pressure results in little to no protein transfer and poor pattern fidelity. For these reasons, a transfer process that can be used to consistently create high-quality micropatterns of isolated islands of dots in just one step is desired.
Herein, a method is described for the indirect micropatterning of islands of micron-sized fluorescent protein adhesion points onto a PAA gel that is more consistent and versatile than previously developed methods. Whereas older indirect micropatterning methods rely on the transfer of protein patterns from a PDMS stamp to an intermediate substrate, the method introduced here uses PDMS stamps instead as a vessel for protein removal, not addition. This is done by first fundamentally changing the structure of the PDMS stamps used. Rather than making stamps that are composed of a pattern of evenly spaced circular pillars, stamps are made up of a pattern of evenly spaced circular holes in this method.
With this new structure, the surface of these PDMS stamps can then be treated with glutaraldehyde as described previously20,29,30, making the stamp able to bond covalently with protein. When used on a glass coverslip evenly coated with fluorescent protein, these glutaraldehyde-treated PDMS stamps are used to remove most of the protein on the surface of the coverslip, leaving behind only the desired pattern of dots predetermined by the location of micron-sized holes on the stamp. This change increases the success rate for generating patterns made up of a near-continuous grid of dots and for creating isolated islands of dots through only one step.

<table><tbody><tr><td>(3-aminopropyl)trimethoxysilane</td><td>Sigma Aldrich</td><td>#281778</td><td></td></tr><tr><td>1.5 mL Microcentrifuge tube</td><td>Fisher Scientific</td><td>#05-408-129</td><td></td></tr><tr><td>15 mL conical tube</td><td>Fisher Scientific</td><td>#05-539-12</td><td></td></tr><tr><td>4 x 4 in 0.060 Quartz LR Chrome Photomask</td><td>Advance Reproductions Corporation</td><td>N/A</td><td>Custom-designed mask</td></tr><tr><td>6 Well Plates</td><td>Fisher Scientific</td><td>#07-200-83</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>#A18P-4</td><td></td></tr><tr><td>Acrylamide Solution, 40%</td><td>Sigma Aldrich</td><td>#A4058</td><td></td></tr><tr><td>AlexaFluor 488</td><td>Thermo Fisher</td><td>#A20000</td><td></td></tr><tr><td>Aminonium Persulfate</td><td>Fisher Scientific</td><td>#BP179-25</td><td></td></tr><tr><td>Bisacrylamide</td><td>Fisher Scientific</td><td>#PR-V3141</td><td></td></tr><tr><td>Ethanol</td><td>Greenfield Global</td><td>#111000200C1GL</td><td></td></tr><tr><td>Glass Coverslips, 25 mm round</td><td>Fisher Scientifc</td><td>#12-545-102</td><td></td></tr><tr><td>Glass Coverslips, 30 mm round</td><td>Warner</td><td>#64-1499</td><td></td></tr><tr><td>Hamamatsu ORCA-R2 Camera</td><td>Hamamatsu</td><td>#C10600-10B</td><td></td></tr><tr><td>Human Plasma</td><td>Valley Biomedical</td><td>#HP1051P</td><td>Used to isolate fibronectin</td></tr><tr><td>Hydrochloric Acid, 1.0 N</td><td>Millipore Sigma</td><td>#1.09057</td><td></td></tr><tr><td>ImageJ</td><td>Wayne Rasband</td><td>#1.53n</td><td></td></tr><tr><td>Interchangeable Coverslip Dish Set</td><td>Bioptechs</td><td>#190310-35</td><td></td></tr><tr><td>Kim Wipes</td><td>Fisher Scientific</td><td>#06-666-11C</td><td></td></tr><tr><td>Mask Alinger</td><td>Karl Suss</td><td>#MA6</td><td></td></tr><tr><td>Matlab 2021</td><td>Mathworks</td><td>#R2021a</td><td></td></tr><tr><td>MetaMorph Basic</td><td>Molecular Devices</td><td>#v7.7.1.0</td><td></td></tr><tr><td>N-hydroxysuccinimide ester</td><td>Sigma Aldrich</td><td>#130672-5G</td><td></td></tr><tr><td>NucBlue Live Cell Stain</td><td>Thermo Fisher</td><td>#R37605</td><td></td></tr><tr><td>Olympus IX2-ZDC Inverted Microscope</td><td>Olympus</td><td>#IX81</td><td></td></tr><tr><td>PD-10 Desalting Columns</td><td>GE Healthcare</td><td>#52-1308-00</td><td></td></tr><tr><td>Photoresist Spinner Hood</td><td>Headway Research</td><td>#PWM32</td><td></td></tr><tr><td>Plasma Cleaner</td><td>Harrick</td><td>#PDC-001</td><td></td></tr><tr><td>Plasma Etcher</td><td>TePla</td><td>#M4L</td><td></td></tr><tr><td>Prior Lumen 200Pro Light Source</td><td>Prior Scientific</td><td>#L200</td><td></td></tr><tr><td>Silicon Wafers, 100 mm</td><td>University Wafer</td><td>#809</td><td></td></tr><tr><td>SU-8 2005</td><td>Kayaku Advanced Materials Inc.</td><td>#NC9463827</td><td></td></tr><tr><td>SU-8 Developer</td><td>Kayaku Advanced Materials Inc.</td><td>#NC9901158</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer</td><td>Essex Brownell</td><td>#DC-184-1.1</td><td></td></tr><tr><td>Tetramethylethylenediamine</td><td>Fisher Scientific</td><td>#BP150-20</td><td></td></tr><tr><td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td><td>Sigma Aldrich</td><td>#448931</td><td></td></tr><tr><td>UAPON-40XW340 Objective</td><td>Olympus</td><td>#N2709300</td><td></td></tr><tr><td>UV Flood Exposure</td><td>Newport</td><td>#69910</td><td></td></tr><tr><td>Wafer Carrier Tray, 110 x 11 mm</td><td>Ted Pella, Inc.</td><td>#19395-40</td><td></td></tr></tbody></table>

pattern generation for micropattern traction microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

PAA hydrogels with the Young&#39;s modulus of E = 3.6 kPa and the Poisson&#39;s ratio of &#957; = 0.445 were made for use by this subtractive micropatterning method. The hydrogels were made to be ~100 &#181;m thick, which allows them to be imaged with the imaging setup used here while also preventing the cells from sensing the rigid coverslip below the gel, which would cause problems in studies focused on cellular rigidity sensing23,33. Gels of ...

Watch this Scientific Journal Video about Pattern Generation for Micropattern Traction Microscopy at JoVE.com

Pattern Generation for Micropattern Traction Microscopy

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

pattern-generation-for-micropattern-traction-microscopy

Research

JoVE Journal

Bioengineering

2.1K Views.  Boston University. We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Video: Pattern Generation for Micropattern Traction Microscopy

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

Engineering

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Developmental Biology

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Biochemistry

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...