Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.
Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.
Over the past few decades, the importance of the mechanical properties of a cell’s environment has garnered increasing recognition in cell biology. Different tissues and extracellular matrices have different relative stiffnesses and, as cells migrate throughout the body, they navigate these changes, using these mechanical properties to guide them1,2,3,4,5,6,7. Cells use the stiffness of a given tissue to inform their motile behavior during processes such as development, wound healing, and cancer metastasis. However, the molecular mechanisms that allow sensation of and response to these mechanical inputs remain largely unknown1,2,3,4,5,6,7.
In order to study the mechanisms through which cells respond to physical environmental cues, the rigidity or stiffness of the substrate underlying adherent cells must be manipulated. In 2000, Chun-Min Lo, Yu-Li Wang and colleagues developed an assay8 whereby an individual cell’s motile response to changing mechanical cues could be directly tested by stretching deformable extracellular matrix (ECM)-coated polyacrylamide hydrogels on which the cells were plated. Cells exhibits a significant preference for migrating towards stiffer substrates, a phenomenon they dubbed “durotaxis.”
Since the original report in 2000, many other techniques have been employed for the study of durotaxis. Steep stiffness gradients have been fabricated by casting gels over rigid features such as polystyrene beads9 or stiff polymer posts10 or by polymerizing the substrate around the edges of a glass coverslips11 to create mechanical ‘step-boundaries’. Alternatively, hydrogels with shallower but fixed stiffness gradients have been fabricated by a variety of methods such as gradients of crosslinker created by microfluidic devices12,13 or side-by-side hydrogel solution droplets of differing stiffness8, or hydrogels with photoreactive crosslinker treated with graded UV light exposure to create a linear stiffness gradient14,15. These techniques have been used to great effect to investigate durotactic cellular movement en masse over time. However, typically these features are fabricated in advance of cell plating and their properties remain consistent over the course of the experiment, relying on random cell movement for sampling of mechanical gradients. None of these techniques are amenable to observation of rapid changes in cellular behavior in response to acute mechanical stimulus.
In order to observe cellular responses to acute changes in the mechanical environment, single cell durotaxis assays offer several advantages. In these assays, individual cells are given an acute, mechanical stimulus by pulling the underlying substrate away from the cell with a glass micropipette, thereby introducing a directional gradient of cell-matrix tension. Changes in the motile behavior, such as speed or direction of migration, are then observed by live-cell phase contrast microscopy. This approach facilitates direct observation of cause and effect relationships between mechanical stimuli and cell migration, as it allows rapid, iterative manipulation of the direction and magnitude of the tension gradient and assessment of consequent cellular responses in real time. Further, this method can also be used to mechanically stimulate cells expressing fluorescent fusion proteins or biosensors to visualize changes in the amount, activity, or subcellular localization of proteins suspected to be involved in mechanosensing and durotaxis.
This technique has been employed by groups who study durotaxis8,16 and is described here as it has been adapted by the Howe Laboratory to study the durotactic behavior of SKOV-3 ovarian cancer cells and the molecular mechanisms that underly durotaxis17. Additionally, a modified method is described for fabrication of hydrogels with a single, even layer of fluorescent microspheres near the cell culture surface; this facilitates visualization and optimization of micropipette-generated strain gradients and may allow assessment of cell contractility by traction force microscopy.
1. Fabrication of Deformable Polyacrylamide Hydrogels with Embedded Fluorescent Microspheres
NOTE: Directions describe polymerization of a 25 kPa hydrogel that is 22 μm in diameter and approximately 66 μm thick. Each or all of these parameters can be modified and directions to do so can be found in Table 1 and in the notes17.
2. Plating cells
3. Preparation of glass micropipette: pipette pulling and forging
4. Positioning the micromanipulator and the micropipette
5. Calibrating the micromanipulator and force generation
6. Conducting the durotaxis assay
7. Determining durotactic migration response
By preparing micropipettes (Figure 1) and normalizing the force generation of the pulls (Figure 2 and Figure 3) as described above, optimal durotactic conditions have been identified for multiple cell lines. Using this technique, as outlined in Figure 4, both SKOV-3 ovarian cancer cells17 and Ref52 rat embryonic fibroblasts (Figure 5) move toward increased stiffness in gradients applied by a glass micropipette. In addition to durotaxis, this method can be used to study dynamic signaling events using fluorescent biosensors and markers. For example, the structure of and signaling within focal adhesion structures can be observed upon durotactic stimulation. Vinculin tension sensor (VinTS) is a FRET-based biosensor which localizes to focal adhesions, allowing for fluorescent observation of focal adhesion dynamics and measurement of changes in tension within those structures19. Ref52 cells transiently expressing VinTS on 125 kPa polyacrylamide gels show the formation of focal adhesions in the direction of stretch over time period of 40 min (Figure 6A). FRET analysis20 reveals that vinculin localized to focal adhesions experiences an immediate change in tension when presented with acute durotactic stimulation (Figure 6B) expanding the utility of this assay to the observation of subcellular signaling events in response to durotactic stimulation.
Figure 1. Diagrams of typical pulled (A) and forged (B) micropipettes. (A) Micropipettes are pulled using a two-step protocol to achieve a taper from 1 mm to 10 μm over 2 mm. (B) Micropipettes are then loaded into the microforge and their tips are bent, enclosed, and shortened so that the last 250 μm of the micropipette is bent at a ~35° angle and tapers from ~30 μm to a rounded tip that measures ~15 μm. Please click here to view a larger version of this figure.
Figure 2. Improved bead field after ethanol coating as compared to traditional method using poly-L-lysine. Representative hydrogel bead fields from poly-L-lysine and ethanol (EtOH) evaporation coverslip coating methods using yellow-green, red, and dark-red fluorescent beads. Scale bar: 25 μm. Please click here to view a larger version of this figure.
Figure 3. Generation of force map for an example durotactic stretch. (A) Position of fluorescent microspheres before and after (pseudo-colored green and red, respectively) deforming the hydrogel with a micropipette (located beyond the right edge of the panel). Scale bar: 25 μm. Displacement vectors (B) and displacement heat map (C) between null and pulled bead fields generated by Traction Force Microscopy plugins in ImageJ highlight the gradient of bead deflection and hydrogel strain. Please click here to view a larger version of this figure.
Figure 4. Schematic of durotaxis assay and determination of deflection angle. (A) A cell is observed for at least 30 min to determine its original trajectory. (B) The micropipette is positioned orthogonally to the cell’s trajectory, 50 μm from the cell edge. The hydrogel is engaged by the micropipette such that moving the micropipette will exert force on the surface of the hydrogel. (C) The micropipette is pulled an additional 20 μm away from the cell, orthogonal to the cell’s trajectory which creates an acute, local gradient of tension (denoted in blue) which increases toward the micropipette. (D) The cell is observed over time as it navigates the applied gradient. (E) In ImageJ or an image analysis program, the original trajectory (dashed line) is marked by a line drawn from the middle of the cell through the center of the leading edge in the first frame. The final trajectory (solid line) is marked by a line drawn after the cell is allowed to navigate the applied tension gradient. The angle between these two lines toward the stimulus is termed “turn angle,” marked here by θ. Please click here to view a larger version of this figure.
Figure 5. Rat Embryonic Fibroblasts move toward regions of increased substrate stiffness in durotaxis. Time course showing durotactic movement of a Ref52 cell 10 min before the pull (panel 1), 1 min before the pull (panel 2), at the time of pull (panel 3), and 1 h after pull (panel 4). Arrow indicates direction of stretch. Scale bar: 50 μm. Please click here to view a larger version of this figure.
Figure 6. Protein localization and activity during durotactic stimulation using fluorescent markers or biosensors. Ref52 cells transiently expressing Vinculin Tension Sensor (VinTS)19 migrating on 125 kPa polyacrylamide gels are presented with acute durotactic stimulation. (A) After stimulation, new focal adhesions form in the direction of stretch as cells re-orient along the stiffness gradient. For two 10 min periods, starting 20 min before mechanical stimulation and 21 min after stimulation, cell morphology (top) and focal adhesion formation (bottom) were monitored. Red color indicates the first timepoint within the time period and green color indicates the timepoint 10 min later. New focal adhesions formed within the 10 min period are shown in green. Before stimulation, new focal adhesions form in the direction of travel. After stimulation, new focal adhesions form in the direction of stretch. Arrow indicates the direction of stretch. Arrowheads indicate areas with focal adhesions formed over that 10 min period. Scale bar: 25 μm. (B) FRET analysis of VinTS fluorescence indicates a change in tension within focal adhesions proximal to durotactic stretch. Outline of cell membrane before and after stretch highlight deformation of cell upon stimulation. Arrowheads indicate examples of focal adhesions experiencing changes in FRET ratio upon stretch. Scale bar: 10 μm. Please click here to view a larger version of this figure.
Desired hydrogel stiffness | 3 kPa | 25 kPa | 125 kPa |
7.5% Acrylamide | 100 μL | 100 μL | 160 μL |
0.5% Bis-Acrylamide | 10 μL | 100 μL | 100 μL |
ddH2O | 287 μL | 197 μL | 137 μL |
Table 1. Acrylamide gel solutions.
Demonstrated here is a repeatable, single-cell durotaxis assay that allows assessment of a cell’s ability to alter its migration behavior in response to acute mechanical cues. This technique can also be used in combination with fluorescence microscopy and appropriate fusion proteins or biosensors to examine subcellular signaling and cytoskeletal events within seconds of mechanical stimulation or over a longer timescale during durotactic movement. Understanding a cell’s relationship to its environment involves the study of the impact of both the chemical and mechanical aspects of that environment. Though potentially difficult to master, this durotaxis assay can be widely used to understand the cellular response to changes in its mechanical microenvironment.
Significance with respect to existing methods
As mentioned before, this micropipette-based method of durotactic stimulation is highly manipulable, allowing a high degree of spatiotemporal control over mechanical stimuli, a major advantage over other techniques, such as pre-formed linear or step-gradients of rigidity. The magnitude and direction of the imparted strain gradients can be visualized by tracking the displacement of fluorescent beads embedded in the hydrogel, near the cell culture surface.
Restricting these fiducial markers to a single layer just below the culture surface increases the accuracy of this tracking. Microspheres located below the plane of deflection (imparted either by the micropipette or, for traction force microscopy, by cellular contractility), as would occur with mixing the microspheres evenly throughout the hydrogel, will move less than in-plane microspheres, which can lead to underestimation of applied forces. Also, this modification is easier to perform and more reliable than methods in which beads are overlaid in an extremely thin layer of polyacrylamide cast on top of a pre-formed gel21 or brought to the hydrogel surface by gravity-assisted settling22 and produces a more even dispersal of beads across the hydrogel than previously described methods17,23.
Modification and future applications
The specifics of this assay can be modified to best suit the cell line of interest. For example, a variety of extracellular matrix molecules (e.g., collagen I, collagen IV, laminin) or other adhesive ligands can be used to functionalize the hydrogel. Also, the starting stiffness of the hydrogel can easily be raised or lowered by tuning the ratio of acrylamide to bis-acrylamide (see Table 1). By changing the dimensions of the micropipette tip and the magnitude of the pull, this assay can be optimized to impart a repeatable and effective durotactic stimulus for the cell type in question.
Critical steps and troubleshooting
Only cells following a steady, linear trajectory of migration prior to hydrogel manipulation should be stimulated to ensure that changes in trajectory are due to the mechanical stimulus and not random fluctuation. Care must be taken to fabricate a glass micropipette that engages the hydrogel surface without slipping but does not tear the gel when pulled. It is important to apply a steady, constant stretch to the hydrogel during the course of the experiment to obtain clean results meaning that the user should be practiced at placing and moving the micropipette before encountering a cell. Any unintentional movement of the micropipette that leads to changes in the tension gradient could affect the cell’s ability to durotax. Similarly, manipulation of the gel should be practiced with each new micropipette that is forged as slight changes in pipet shape can cause the pipet/gel interaction to vary.
Failure to position the micropipette within the microscopic field of view before increasing magnification can lead to the accidental breakage of the fragile glass tip. Ensure that the height and X-Y position of the tip is known before lowering the micropipette with the micromanipulator. Always monitor the position of the micropipette to reduce the risk of breakage. It is recommended that the magnification is decreased back down to 10X for each new micropipette loaded into the micromanipulator as slight movements of the micromanipulator and pipet sheath can lead to large apparent changes in the position of the newly loaded micropipette.
Before finding cells to observe, it is important to first test the micropipette to confirm that it will engage the hydrogel as expected and that it is suitable for applying the desired stretch. Finding tip dimensions that suit the experiment and cell type is critical to success in applying durotactic stimulation. The end of the micropipette should be rounded enough so that it does not break through the gel, but not so rounded that it fails to grip it. If the pipet does not pull gel effectively, it may be sliding along the surface. The shape of the micropipette tip may be too rounded to properly engage with the gel surface. The dimensions at the very tip of the micropipette should be adjusted until firm, steady contact can be achieved consistently. In some cases where the micropipette is slipping across the gel surface, it may be necessary to lower the micropipette further into the hydrogel to gain more traction. If the micropipette tears through gel, the tip may be too fine or too sharp. Gel tearing may also indicate too much force is being applied while pulling. The micropipette should be raised slightly to reduce gel deformation and pulling shorter distances.
Often, if the cell or the edge of the cell is pulled out of focus by the micropipette, the tip of the micropipette is engaged too close to the cell or the stretch is too forceful. Move the micropipette further from the cell, only slightly deforming the cell in the X-Y planes. Moving the cell out of focus will not only make cellular events impossible to monitor and cause optical aberrations, but it will cause the cell to experience more stimulation than the 2-dimensional tension gradient intended.
Most importantly, it is critical to record and analyze only responses that have consistent durotactic manipulation with minimal human error. If the lowering of the tip is imprecise or if the micropipette is repositioned, the results of the experiment will be clouded. Since this assay is complex and many steps are prone to error, care must be taken at every step to avoid unintentional changes in the stimulation of cells. Failure at any step can lead to inconsistent stretch application and unreliable results.
Limitations
There are limitations to this technique that should be considered. Most prominently, accurate forging and manipulation of the glass micropipettes can present a steep learning curve for new users. Additionally, the position and magnitude of the hydrogel pull must be optimized for different cell lines. Examining fluorescent bead displacements before and after hydrogel manipulation can help with this aspect of the technique. Also, while the technique allows high spatiotemporal observation of durotactic behavior in individual cells, this makes it a low-throughput assay. It is therefore important to point out that this assay can also be complemented by other techniques with lower manipulability but higher throughput, such as using hydrogels with pre-formed gradients of rigidity, to analyze the durotactic behavior of larger populations of cells at once. In summary, the high degree of spatiotemporal control of mechanical cues afforded by the single-cell durotactic assay make it very useful for parsing the molecular mechanisms contributing to the durotactic behavior of many different cell types under many conditions.
The authors have nothing to disclose.
None.
Acrylamide 40 % | National Diagnostic | EC-810 | |
Ammonium Persulfate | Fisher | BP179-25 | |
BD20A High frequency generator | Electro Technic Products | 12011A | 115 V - Handheld Corona Wand |
Bind Silane (y-methacryloxypropyltrimethoxysilane) ( | Sigma Aldrich | M6514 | |
Bis-acrylamide 2% | National Diagnostic | EC-820 | |
Borosilicate glass capillaries | World Precision Instruments | 1B100-4 | |
Branson 2510 Ultrasonic Cleaner | Bransonic | 40 kHz frequency | |
Coarse Manipulator | Narshige | MC35A | |
DMEM | Corning | 10-013-CV | |
DMEM without phenol red | Sigma Aldrich | D5030 | |
Dual-Stage Glass Micropipette Puller | Narshige | PC-10 | |
Epidermal Growth Factor | Peprotech | AF-100-15 | |
Ethanol | Pharmco-aaper | 111000200 | |
Fetal Bovine Serum (Qualified One Shot) | Gibco | A31606-02 | |
Fibronectin | EMD Millipore | FC010 | |
Fluospheres Carboxylate 0.2 um | Invitrogen | F8810, F8807, F8811 | |
Fugene 6 | Roche | 1815091 | 1.5 ug DNA / 6uL fugene 6 per 35mm dish |
Glacial Acetic Acid | Fisher Chemical | A38SI-212 | |
Glass Bottom Dish | CellVis | D60-60-1.5-N | |
Glass Coverslip | Electron Microscopy Sciences | 72224-01 | 22 mm, #1.5 |
HCl | JT Baker | 9535-03 | |
Hellmanex III Special cleaning concentrate | Sigma Aldrich | Z805939 | Used at 2% in ddH2O for cleaning coverslips |
HEPES powder | Sigma Aldrich | H3375 | Make 50mM HEPES buffer, pH 8.5 |
Intelli-Ray 400 Shuttered UV Flood Light | Uviton International | UV0338 | |
Isopropanol | Fisher Chemical | A417-4 | |
Microforge | Narshige | MF900 | |
Micromanipulator | Narshige | MHW3 | |
Mineral Oil | Sigma Aldrich | M5904 | |
Nanopure Life Science UV/UF System | Barnstead | D11931 | ddH2O |
Nikon Eclipse Ti | Nikon | ||
OptiMEM | Invitrogen | 31985062 | |
Parafilm M | Bemis Company, Inc | PM-992 | |
PBS | 139 mM NaCl, 2.5 mM KCl, 28.6 mM Na2HPO4, 1.6 mM KH2PO4, pH 7.4 | ||
Platelet Derived Growth Factor-BB (PDGF-BB) | Sigma Aldrich | P4056 | |
Ref52 | Rat embryonic fibroblast cell line; Culture in DMEM + 10% FBS | ||
Ringer's Buffer | 134 mM NaCl, 5.4 mM KCl, 1 mM MgSO4, 2.4 mM CaCl2, 20 mM HEPES, 5 mM D-Glucose, pH 7.4 | ||
SKOV-3 | American Type Culture Collection | Culture in DMEM + 10% FBS | |
Sulfo-SANPAH | Covachem | 12414-1 | |
Tabletop Plasma Cleaner | Harrick Plasma | PDC-32G | |
TEMED | Sigma Aldrich | T9281-50 |