D Confocal microscopy image of hydrogel patterned with a custom design inset: binary image and reacted with NHS-AF dye. E Confocal microscopy image of hydrogel patterned with a virtual mask exhibiting a linear gradient in gray values and reacted with NHS-AF dye; the inset shows the fluorescence intensity profile along the white dahsed line.
The inset shows the locations of each point overlaid on an epifluorescence image of the hydrogel surface. The values obtained for points 6—10, which are within the pattern, show no difference compared to the ones outside the pattern. Figure 3. Pattern functionalization on hydrogels via different chemistries. Figure 4. Cell patterning on poly acrylamide hydrogels. Patterns were functionalized with indicated integrin ligands.
A Confocal microscopy image of fixed and phalloidin-stained pHDF cells. Phalloidin stains filamentous actin; nuclei are stained in blue. D Phase contrast microscopy image of live pHDF recognizing line patterns of fibronectin immobilized through biotin—streptavidin chemistry.
Figure 5. Size of cell adhesive patterns on hydrogels affects cell mechanosensing. Each data point corresponds to a single cell, from two independent experiments. Figure 6. Applicability of patterned poly acrylamide hydrogels for traction force microscopy applications. A Confocal microscopy image of fluorescent beads on the upper layer of the hydrogel, merged with the transmission image showing a live pHDF fibroblast adhering on a line pattern schematically overlaid in gray.
B Particle image velocimetry PIV analysis results from bead displacements observed before and after the removal of the cell shown in A. The magnitude of the color-coded vectors is given in pixels. C Confocal microscopy image of a patterned and AlexaFluor labeled hydrogel. Cell outlines white in D, E are a guide to the eye. Hydrogels were washed three times for 5 min with PBS prior to cell seeding. Hydrogels were first reacted with 4. Hydrogels were washed five times for 5 min with water prior to imaging, or three times for 5 min with water and two times for 5 min with PBS prior to cell seeding.
Then, a click reaction with an alkyne-coupled AlexaFluor dye 0. Hydrogels were washed five times for 5 min with water prior to imaging. Hydrogels were first reacted with 2. Such files may be downloaded by article for research use if there is a public use license linked to the relevant article, that license may permit other uses. The authors thank Prof. The Max Planck Society is appreciated for its general support.
More by Dimitris Missirlis. More by Felix Lussier. More by Joachim P. Cite this: ACS Appl. Published by American Chemical Society. Article Views Altmetric -. Abstract High Resolution Image.
Understanding how cells sense and respond to the physical and mechanical properties of their insoluble microenvironment, i. Among different approaches, the ex vivo interrogation of cells on artificial substrates with controlled biophysical and biochemical properties has proven to be a powerful tool to test hypotheses and gain mechanistic insight into mechanosensing and mechanotransduction of living cells.
Upon adhesion on a compliant substrate, cells exert traction forces at the sites of attachment, where multiprotein complexes, termed focal adhesions FAs , assemble. An attractive approach to achieve this goal is to pattern adhesive ligands on viscoelastic substrates so that cells conform to the designed patterns. Hydrogels based on synthetic or natural non-ECM polymers offer the advantage of decoupling ligand presentation from mechanical properties, due to their tunable stiffness and biologically inert background, on which adhesive ligands can be incorporated.
The first, most common strategy is based on localized activation of reactive groups or exposure of protein-adsorbing surfaces on the gel surface and subsequent, selective attachment of ligands. Among the developed hydrogel systems, polyacrylamide pAAm gels remain the most popular choice for cell mechanobiology studies due to their ease of fabrication and established use in traction force microscopy TFM studies following decoration with fiducial markers.
Consequently, there is room for improved techniques that are more versatile, easy-to-perform, and accessible. Here, we introduce a facile and versatile method to pattern pAAm hydrogels using UV light, through the co-polymerization of monomers containing light-sensitive caged amine groups. The generated primary amines can be subsequently used to immobilize cell ligands throughout various conjugation methodologies.
The presented technique takes advantage of a commercially available micropatterning system on an optical microscope but should be applicable on most standard laser-based microscopy systems of a typical research lab. Results and Discussion. Poly acrylamide pAAm hydrogels were prepared via radical cross-linking polymerization of acrylamide Am and bis-acrylamide Bis Figure S1.
The Am and Bis concentrations determine the cross-linking extent, and hence the stiffness of the resulting pAAm hydrogels. To include reactive groups in the polymer network, the introduction of appropriate comonomers in the polymerization mixture has been proposed.
On the other hand, the incorporation of 2-aminoethylmethacrylamide AEMA at concentrations between 0.
The presence of nucleophilic primary amines in AEMA-containing hydrogels was quantified using fluorescamine, and the amount of incorporated comonomer increased linearly with precursor concentration, providing control over the number of incorporated reactive groups Figure 1 B. High Resolution Image. To introduce amines in a spatially controlled manner, a light-sensitive comonomer was synthesized and incorporated in the hydrogels.
Moreover, preliminary studies showed that a higher amount of radical initiation was required for polymerization due to the radical scavenging properties of the nitro group. The mechanical properties of the resulting hydrogels were influenced by the presence of the caged comonomer, depending on the cross-linking ratio: for the stiffer hydrogels 0.
For the lower cross-linking concentration 0. The caged comonomer was designed such that the cleavage of the carbamate group upon UV irradiation would expose primary amines for further functionalization as schematically shown in Figure 2 A. The plateau value was the same as the one for hydrogels containing AEMA at the same concentration, indicating complete uncaging of caged AEMA at illuminated regions. Nonpatterned areas exhibited similar fluorescence to the negative controls no AEMA incorporated , demonstrating a lack of uncaging from ambient light in nonilluminated regions.
The PRIMO micropatterning system with its associated software also enabled the facile preparation of virtual masks, and thus the patterning of custom designs Figure 2 D , as well as the formation of gradients of immobilized fluorescent labels, through the use of virtual masks with a gradient in gray value Figure 2 E. Patterning of two different dyes in complex, predesigned patterns was also achieved with sequential light exposure, fluorescent labeling, and alignment using the fluorescence of the first pattern Figure 2 F.
In principle, localized patterning with the hydrogels presented here is also feasible in the z -direction using two-photon microscopy. Having validated the presence of reactive amines on patterned regions Figure 2 , three different chemistries were examined with the aim of introducing spatially patterned functional molecules on the surface of poly acrylamide hydrogels Figure 3.
First, deprotected amines in hydrogels were reacted with a heterobifunctional NHS-biotin linker to exploit the robust biotin—streptavidin interaction to immobilize ligands on surfaces.
In this case, AttoStreptavidin was only present on the hydrogel surface, due to the size-dependent exclusion of the protein from diffusion inside the hydrogel Figure S2. Second, click chemistry was performed following reaction of patterned hydrogels with a heterobifunctional NHS-PEG 5 -Alkyne linker: an azide-functionalized fluorophore AlexaFluor was used to validate this approach Figure 3 B.
Inversely, the azide was immobilized on the patterned hydrogel using a heterobifunctional NHS-PEG 4 -azide linker and patterned regions were labeled with alkyne-functionalized AlexaFluor Figure 3 C. Due to maleimide degradation in aqueous solutions, these two steps should be performed in quick succession. The above results highlight the versatility of patterning this type of hydrogels with commonly used bioconjugation chemical strategies.
An unmet challenge for the majority of existing patterning methods for pAAm hydrogels is the patterned immobilization of short peptide or peptidomimetic ligands. Techniques to pattern such low-molecular-weight ligands have been reported for other hydrogel systems; 23,44,45 here, we demonstrated as a proof of principle, the patterning in lines of integrin peptide or peptidomimetic ligands on pAAm hydrogels, and visualized cell adhesion using optical microscopy.
Small peptidomimetic integrin-selective ligands 46,47 were also immobilized using the maleimide-thiol coupling strategy. Immobilization of larger ECM proteins, such as fibronectin FN , was also successful using the biotin-avidin linkage to confine cells in defined patterns Figure 4 D. The above results demonstrate the versatility of the presented system to pattern various ligands and the generality in application through the use of different cell types.
We next examined how the shape of micropatterned ligands affects cell mechanosensing. Notably, the cell size distribution was narrower on patterned substrates compared to the homogeneously coated hydrogel, demonstrating an advantage of cell patterning Figure 5 B. Next, the localization of yes-associated protein 1 YAP , a mechanosensitive transcriptional regulator that shuttles between the nucleus and cytoplasm depending on cell shape and actomyosin contractility, 48 was examined.
Previous work has demonstrated that confinement of cell size by reducing accessible adhesive area on rigid glass, or culturing of cells on soft substrates, leads to YAP nuclear export. Fibroblasts adhering to homogeneously FN-coated hydrogels of the same stiffness showed mostly nuclear YAP localization as expected 49,50 Figure 5 C,D , highlighting the effect of pattern shape on mechanotransduction.
In sum, the above results demonstrate how control over the size of adhesive patterns on soft elastic hydrogels can be used to study cell behavior and open the way for studying the combinatorial effects of substrate stiffness, cell shape, and ligand type.
Of particular interest is the investigation of the relative contributions of substrate stiffness and cell area on YAP mechanotransduction to test recently developed models. One major application of pAAm hydrogels remains traction force microscopy TFM for the estimation of cell-generated forces, 34 despite the development of more physiologically relevant viscoelastic substrates or three-dimensional 3D culture systems.
In TFM, fluorescent beads embedded in the elastic substrate serve as fiducial markers that report on substrate deformations induced by cells. The confinement of cells within adhesive patterns on top of TFM substrates allows the estimation of traction forces as a function of cell shape, as well as the averaging over many cells to obtain more reliable conclusions.
The photopatterning process did not cause bleaching of the beads and pHDF fibroblasts spread and migrated along the patterned lines Figure 6 A. Importantly, standard protocols could be used to determine substrate deformations through particle image velocimetry PIV analysis Figure 6 B. Time lapse imaging of cells moving on patterns showed how deformations are spatially confined in the axis of the patterned lines and can be used for future calculations of exerted traction forces Movie S2.
Traction force determination requires the reference relaxed state of the hydrogel, which is typically achieved following cell detachment. An alternative approach is to perform reference-free traction force microscopy using a regular pattern, which upon deformation, and hence deviation from the ideal mesh, can inform on the forces exerted on the substrate. Paxillin is a marker of focal adhesions in cells.
An analogous analysis as for the standard TFM substrates was performed Figure 6 D,E and Movie S3 , demonstrating the applicability of this approach to measure substrate deformations. The micropatterning strategy introduced here provides a versatile method to introduce cell adhesive ligands on demand, without the need to create photomasks or use harsh reaction conditions.
The approach uses mild UV light to uncage reactive groups within the cross-linked polymer network; subsequent functionalization with established chemistries was demonstrated here for commonly used bioconjugation methods but can be expanded to other chemistries.
Importantly, this methodology allows the patterning of low-molecular-weight ligands, a challenge with existing methods, which mostly relied on nonspecific reaction with amino acid side chains of ECM proteins. The applicability of the patterned substrates was exemplified by demonstrating the regulation of YAP activity as a function of pattern size on physiologically stiff substrates. The presented hydrogels are compatible with standard TFM protocols and could potentially be adapted for use in reference-free TFM.
Experimental Section. A list of commercially available reagents and antibodies used in this study are presented in the Supporting Information Tables S1 and S2.
Then, an equimolar amount of 4,5-dimethoxynitrobenzyl chloroformate After stirring at room temperature for a period of 1 h, the reaction was diluted with 15 mL of dichloromethane, followed by the addition of 10 mL of a 1 M KHSO 4 for acidification. Poly acrylamide hydrogels were prepared using radical polymerization according to a published protocol. Comonomers were introduced in the precursor solution at defined concentrations.
Fluorescent beads, nm in diameter, were added stock solution to the precursor mixture prior to gelation. The hydrogel thickness was controlled using spacers between the two coverslips. Gelation was left to proceed for at least 30 min at room temperature. Gels attached to the APTES-treated coverslips were recovered after removing the hydrophobic coverslip and were washed in excess of water five times to remove unreacted monomers and initiators.
The exact value of the spring constant was determined using the thermal noise calibration method prior to measurements. Force—distance F—d curves were obtained from immobilized gels with a cantilever speed of 1. A Poisson ratio of 0. At least 30 curves from at least three different positions were analyzed per hydrogel. The amount of incorporated AEMA was determined using the amine-reactive dye fluorescamine.
The well plate was shaken for 10 min, and fluorescence intensity was measured using a TECAN well plate reader excitation wavelength: nm; emission wavelength: nm. AEMA concentrations were calculated using a calibration curve obtained from aqueous comonomer solutions. Precursor solutions without the comonomer were used as blanks. Patterns virtual masks were designed using the software Inkscape and were saved as 8-bit Tiff files levels of gray value.
The designed patterns were then loaded on the Leonardo software, the objective was focused on the hydrogel surface, and the UV dose was selected. After illumination, hydrogels were washed five times for 5 min with water prior to functionalization and were protected from light. Micropatterned hydrogels were equilibrated in 10 mM PBS prior to the reaction of deprotected primary amines with heterobifunctional linkers.
Four strategies to functionalize the hydrogels were performed. The hydrogel was then irradiated with a portable UV light lamp for 5 min and rinsed first with water and then with PBS. Hydrogels were washed three times with PBS prior to cell seeding. Ginsberg UCSD. Geiger Weizmann Institute. Cell cultures were checked regularly for the absence of mycoplasma.
After washing with PBS, the cells were permeabilized with 0. Epifluorescence and phase contrast microscopy experiments were performed on a Leica DMi8. Supporting Information. Author Information.
Joachim P. The authors declare no competing financial interest. Integrins as Biomechanical Sensors of the Microenvironment. Cell Biol. Nature Research. Integrins, and integrin-mediated adhesions, have long been recognized to provide the main mol. Recent evidence has shown that their combined biochem. Here, we review this work first by providing an overview of how integrin function is regulated from both a biochem.
Then, we address how this biomech. Finally, we discuss the importance of this sensing for major cell functions by taking cell migration and cancer as examples. Annual Reviews. A review. It is increasingly clear that mechanotransduction pathways play important roles in regulating fundamental cellular functions. Of the basic mech. Cells typically use many mechanosensitive steps and different cell states to achieve a polarized shape through repeated testing of the microenvironment.
Indeed, morphol. Patterned substrates and controlled environments with defined rigidities limit the range of cell behavior and influence cell state decisions and are thus very useful for studying these steps.
The recently defined rigidity sensing process provides a good example of how cells repeatedly test their microenvironment and is also linked to cancer.
In general, aberrant extracellular matrix mechanosensing is assocd. Hence, detailed descriptions of the steps involved in sensing and responding to the microenvironment are needed to better understand both the mechanisms of tissue homeostasis and the pathomechanisms of human disease. Integrins are the major family of adhesion mols. They are essential for embryonic development and influence numerous diseases, including inflammation, cancer cell invasion and metastasis.
In this Perspective, we discuss the current understanding of how talin, kindlin and mech. Biomaterials , 32 , — , DOI: Elsevier Ltd. The interplay between the generation of, and response to, mech. We postulate that adherent cells respond to a no. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios.
Contrary to previous findings, we find that traction force is not detd. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry.
We propose that the responses of traction forces to center-periphery distance, possibly through a pos. A similar pos. Cell Motil. Cytoskeleton , 63 , — , DOI: Wiley-Liss, Inc. Cells display a large variety of shapes when plated in classical culture conditions despite their belonging to a common cell type.
These shapes are transitory, since cells permanently disassemble and reassemble their cytoskeleton while moving. Adhesive micropatterns are commonly used to confine cell shape within a given geometry.
In addn. Modulation of the pattern geometry allows the anal. In this study, we show that the acquisition of cell shape follows two stages where initially the cell forms contact with the micropattern.
Here, the most distal contacts made by the cell with the micropattern define the apices of the cell shape. Then secondly, the cell borders that link two apices move so as to minimize the distance between the two apices. In these cell borders, the absence of an underlying adhesive substrate is overcome by stress fibers forming between the apices, which in turn are marked by an accumulation of focal adhesions.
By inhibiting myosin function, cell borders on nonadhesive zones become more concave, suggesting that the stress fibers work against the membrane tension in the cell border. Moreover, this suggested that traction forces are unevenly distributed in stationary, nonmigrating, cells.
By comparing the stress fibers in cells with one, two, or three nonadherent cell borders it was reasoned that stress fiber strength is inversely proportional to no.
We conclude that cells of a given area can generate the same total sum of tractional forces but that these fractional forces are differently spaced depending on the spatial distribution of its adherence contacts. Geometric Control of Cell Life and Death. Science , , — , DOI: Chen, Christopher S. Science Washington, D. American Association for the Advancement of Science.
Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension.
Cell spreading also was varied while maintaining the total cell-matrix contact area const. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion.
Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment. Nature , , — , DOI: Nature Publishing Group. The student will be expected to apply skills learned in previous courses—Fundamentals of Omega and Practical Time Processing Land or Marine. The student will also have a number of sessions with an area geophysicist who will offer geophysical advice and discuss the proposed processing flows, parameters, and results.
Having sent the customer the required deliverables, the student will make a final presentation detailing their chosen processing flow to a panel of experienced staff on the last day of the course. The course is intended for processing group members wishing to progress to the role of group or project leader.
At the end of the class, students will have developed the functional hands-on practical skills needed to be a contributing member of a depth imaging production processing team. These skills will cover a common simple workflow involved in processing a marine 3D dataset through a depth imaging sequence. The class is suitable either for less experienced geophysicists wanting to learn the basics of depth imaging and for experienced depth imagers wishing to secure the functional capabilities necessary to perform depth imaging.
This five-day hands-on course introduces the geoscientist to the functionality of the WesternGeco Petrel plug-ins that are part of the Omega Earth Model Building bundle. This is primarily a software course where students will use practical exercises to explore the earth model building workflow. This class is suitable for geophysicists with a good knowledge of the Omega platform wanting to gain a practical overview of the earth model building workflow.
This three-day hands-on course introduces the geoscientist to the functionality of the WesternGeco Petrel Gravity and Magnetic plug-in. This is primarily a software course where students will use practical exercises to explore the gravity and magnetic domain preconditioning and anomaly enhancement and modeling workflows.
This class is suitable for geophysicists with a basic knowledge of the Petrel platform and gravity and magnetics geophysical methods wanting to gain a practical overview of the tools to extract information from data and integrate it with geological knowledge, seismic data, and well logs.
This course introduces the physical concepts of each electromagnetic measurement and further illustrates the added value of integrating these methodologies to better understand specific geologic targets in both marine and land environments.
This course also introduces the features of the Petrel Magnetotellurics and Petrel Controlled-Source EM plug-ins, including display, pre-conditioning, QC, modeling, and interpretation of electromagnetics data.
The explanation of the software features is interleaved with practical exercises to allow the attendees practicing with the Petrel plug-ins. Although the course focus is centered on practical examples, theory of physical principles of each electromagnetic discipline along with a basic introduction to the data acquisition process will be discussed.
Geophysicists, geologists, and geoscientists working with electromagnetic magnetotelluric or controlled-source electromagnetic data. Description The student will learn how to execute each of the following Petrel Quantitative Interpretation plug-in workflows—rock physics, AVO modeling, AVO reconnaissance, simultaneous inversion, and stochastic inversion.
Each topic is introduced with some background material, including discussion of theory and applications. This is followed by an explanation of the workflow and data requirements and a step-by-step discussion of the procedures to execute the workflows. In each case, there are exercises to provide hands-on experience in executing the workflows. The training emphasizes the links between the different workflows and how one enables the other. This course is designed for experienced geoscientists involved in exploration, development, and production activities who wish to learn how to use the Petrel Quantitative Interpretation plug-in workflows listed above.
This three-day hands-on training class provides students with the foundations needed to use the Petrel RTI plug-in tools in a full range of acquisition quality control, survey design, and imaging tasks. The course teaches how to create surveys for the different acquisition configurations, how to trace rays using different methods, create synthetic data, illuminate targets, and apply the results in seismic survey design, processing, and imaging workflows. This class is suitable for geophysicists and geologists wanting to learn how to integrate the Petrel RTI plug-in tools into acquisition QC, survey design, and imaging workflows.
This two-day hands-on training class provides students with the skills needed to use the Petrel SSD plug-in in a full range of quality control and design tasks for land, vertical seismic processing VSP , and marine seismic acquisition planning from field QC to advanced design analyses. The class teaches how to create surveys for different acquisition configurations marine, land, and coil , perform fold analyses, and integrate the SSD plug-in with the Petrel Earth Model Building EMB bundle.
Privacy Terms and Conditions Sitemap. Unlock your geophysics data potential with the industry's most advanced geophysical processing software solution Request More Info Software Support. Learn more. Flexibility Choose from more than seismic function modules SFMs to create the optimal workflow for your data, and use a software development kit to build new SFMs with your own algorithms.
Interactivity Easily and intuitively manage seismic projects and data, build workflows, QC processing results, visualize and analyze data, and interactively generate statics and velocity solutions.
Automation Concentrate on geophysics thanks to a database-driven project model, automated data management, job submission, and detailed history. To learn more, explore the links or request more information. Image Viewer. The VISTA IVA application for the Omega platform is a sophisticated, interactive velocity analysis tool bringing versatility and efficiency to the velocity analysis workflow in the time-domain.
Analyze and compare seismic data using the SeisView application in Omega. Omega Seisflow is used to build the complex workflows needed to creatively address unique geophysical challenges. Processing workflows are created interactively by selecting and inserting one of more than algorithms into the Omega Seisflow canvas. Create displays using attribute data files, seismic data files, text, and image files.
Shown: Interactive first-break picking. See What's New in Omega Explore the latest innovations of our geophysical data processing platform. Request More Info. Training Training Program NExT offers a comprehensive training program to support users of the Omega geophysical data processing platform, WesternGeco plug-ins, and other software products.
Consultancy Are you looking for expert geophysical guidance and advice on your unique technical challenges? Training Courses Omega: Fundamentals of Omega 5 Days Description This course will provide knowledge and understanding of the Omega platform processing package from setting up a project through use of the key applications.
View schedule, register, and learn more Omega: 4D Seismic Processing with Omega 1 Day Description During this course, the user will learn how to run 4D diagnostics on a time-lapse dataset at various stages of seismic processing. Who Should Attend This class is suitable for geophysicists involved in 4D processing, interpretation, or both. Omega: Omega Administration 5 Days Description This class introduces the administration tasks required for maintaining an Omega platform installation.
Who Should Attend This class is suitable for system administrators wanting to learn how to install and maintain an Omega platform processing system. Network engineering and integration capabilities include design and analysis, validation and verification, logistics and operations and business process re-engineering BPR. Our innovative engineers bridge traditional engineering with varied electronic, human and cultural elements to advance and agency's overall mission objectives.
We provide fully outsourced management of networks, IP telephony, messages and call centers, virtual private networks VPN , web hosting, video networking and other important IT services. We establish and use systematic, disciplined and quantifiable engineering approaches and best practices to develop economical relable and functional software.
Welcome to KBR.
0コメント