Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Biological tissues grow in three-dimensional soft environments through which cells are exposed to various mechanical cues. Our method allows the stretching of hydrogels in a three-dimensional manner for the investigation of biomechanical responses. This technique enables the uniform stretching of hydrogels along their thickness while live imaging is performed.

Additionally, the hydrogel’s geometry can be manipulated to any size or shape. Knowledge about how cells and extracellular matrix respond to forces is important for revealing how tissues develop and diseases progress. This can lead to potential development of cancer therapies and tissue engineering models.

Begin by using a laser or manual cutter to create a rubber strip with a hole in the center. Cut a rectangle of hydrophobic film so that it is wider than the silicone strips and wash a plastic dish with 70%ethanol. After drying with lint-free wipes, place the sealing film into the dish and place one silicone strip with the plastic wrap removed from one side into the center of each piece of sealing film.

For fibrin gel formation, uniformly deliver 2.5 microliters of cold labeled fibrinogen into the silicone cutout of each strip so that the entire circumference of each cutout is in contact with fibrinogen, taking care not to allow any air pockets or bubbles to form anywhere in the solutions. Immediately add 2.5 microliters of cooled thrombin directly to each fibrinogen solution and quickly mix the solutions with careful pipetting, moving the tip around the entire mixture to create as homogenous a solution as possible. After mixing, place the covered dish in the incubator for 30 minutes for gel polymerization.

At the end of the incubation, add enough PBS to the dish to submerge the gel silicone constructs and carefully lift each sample from the dish, making sure that the sealing film layer remains adhered to the strip. Slowly peeling from one end of the silicone to the other, carefully detach the sealing film from each piece of silicone, then place the strip back into the dish and place the dish on the stage of a light microscope to assess the condition of the sample. To load the sample onto the stretching device, fill the bath with PBS and place the silicone strip containing the sample gel across the top of the bath so the ends of the strip are sitting on each side of the bath.

Place the clamps and fabric strips, and then tighten so that all of the pieces are connected to form one straight strip with the cutout in the center. Place the material on the well and secure a cover slip on the bottom of the well, then place an aluminum liquid well into the stretching device. Fill the well with one to two milliliters of PBS cell medium and place the strip fabric gel construct into the device.

Clamp the fabric strip into the bracket so that the cutout of the gel is in the center before carefully placing and locking the pin down insert into place in the device. Place and lock other fabric side into the spindle and place the stretching device and the attached sample onto the stage of a confocal microscope. Use a USB cable to connect the microcontroller to the computer and connect the servo motor to the microcontroller.

Then open the stretching device control module on the computer to image the sample. To determine whether the sample is adequate for an experiment, use low resolution live imaging to scan the entire gel and determine the lowest Z position at which full adhesion to the inner walls of the cutout is apparent with no tears or bubbles. After noting the Z location of the microscope, scan the interface of the fluorescent label of the gel and silicone strip to determine the full adhesion of the gel to the silicone throughout its circumference.

After scanning, move the stage in the Z direction until there is no longer continuity in the gel and note the upper limit of the Z position. Then subtract the upper limit of the Z direction from the lower limit to calculate the sample thickness. To determine the pre-stretch position of the sample, click Go to Zero Servo POS in the module software to make sure the servo motor is at its zero position and attach the servo motor to the stretching device, taking care and not to put any strain on the sample.

While imaging, click the plus one button to move the motor one degree at a time in the clockwise direction. When the right side of the cutout moves, click the minus one button to reverse the sample to the penultimate position to maintain the sample under minimal tension. Click Set Min Servo Position to set the reference position and capture a 40 time magnification, high resolution, single Z slice tile image of the entire gel area as a baseline reference for the post-processing analysis.

When all of the images have been acquired, advance the servo motor one degree per second until the desired stretch magnitude is reached. At each stretch magnitude for which an analysis is desired, verify that the gel has not detached from the silicone at any point throughout its circumference by scanning the interface between the gel and the silicone, then capture a high resolution tile Z stack image set of the entire gel area for post-processing analysis. Zooming in and manually tracking bead aggregates during gel stretching allows calculation of the local gel strains in the axial and perpendicular directions.

Typically the axial strands propagate relatively linearly from the silicone cutout edge to the center of the gel and are larger than the compressive perpendicular strains. Here, high magnification images of a typical unstretched and relatively isotropic hydrogel, and a hydrogen under high 80%cutout strain depicting highly aligned fibers in the stretch direction are shown. Analysis of the fiber reorientation revealed an approximately linear dependence between the fiber alignment and the external strain on the cutout up to strains of 40%with moderate saturation at strains above 40%As illustrated in this fiber alignment analysis, as the external strain on the cutout increases, the fiber alignment increases, following an overall non-linear curve.

Note that the gel response to stretch is relatively uniform along its Z thickness. As illustrated in this preliminary finite element simulation performed on 2D continuous material, the color mat ranges from 38 to 42%indicating that the gel strains are relatively homogenous throughout the circular gel area. It is important to ensure the hydrogel completely adheres to the inner walls of the geometric cutout, as adhesion and continuity is crucial for reliable stretching of samples.

By modifying the geometry of the silicone cutout, we can program gradients in strain and fiber alignment, so when embedding cells we can analyze the response to these gradients.