Our research focuses on utilizing the unique properties of granular hydrogels to investigate cell movement and functions for tissue regeneration. Cell response to the microenvironment is important to model in understanding the implications for translational impact. As the use of 3D port scaffolds increases, so does the need for comprehensive 3D migration assays that better replicate cell behavior in a wound healing environment.
We have developed two pipelines that are three-dimensional, from scaffold development to imaging and analysis, for investigating cell movement and migration in vitro. With these two new assays, we have gained the ability to track the responsive cells at two distinct stages of wound healing:initial scaffold infiltration from bulk tissue and cell movement once surrounded by a complex scaffold. To begin, plate 120, 000 human dermal fibroblasts cells per square centimeter in at least six wells of a 96-well plate.
After overnight incubation, remove the media from the cell wells using an aspirator or pipette. Add the dye in 20 microliters of each gel condition to the wells using a positive displacement pipette. Using a plate spinning rotor centrifuge attachment set at 25 degrees Celsius, spin the plate at 100g for 15 seconds with acceleration and deceleration set to 8 to flatten the gel.
Flip the plate 180 degrees and spin again to ensure even gel distribution across the well bottom. To aseptically photo crosslink the gel, apply focused light at 365 nanometers for 30 seconds to anneal the scaffold. After forming all scaffolds, add 200 microliters of media to each well and incubate at 37 degrees Celsius for 30 minutes.
Use a confocal microscope to image the cells and capture their migration behavior. For image analysis, open the Imaris Image Conversion Software for batch conversions. Drag and drop microscopy images into the conversion software and select a folder within the software arena to import the files.
Select each file individually to set the Voxel Size. Press Start All to begin the conversion. Next, in the Imaris Arena Software, select an image from the arena to begin processing.
Click on the Image Pros tab in the main toolbar. Now, click the dropdown menu for Channel 1 and select Background Subtraction. Press OK to return to the 3D view.
In the small toolbar, click the icon with rounded blue shapes labeled Add New Surfaces to create an editable objects tab named Surfaces 1. Manually generate parameters for all replicates using the blue arrow button. Set the surface detail to 0.7 micrometers and select Background Subtraction, Local Contrast.
Enter the average cell length into the diameter of the largest sphere, which fits into the object box. For thresholding, determine the intensity histogram to segment only the brightest cells. Enable Split Touching Objects, Region Growing, and set the seed points diameter to match the previously used diameter.
To save the creation parameters for batch analysis, click the wand icon labeled Creation. Click store parameters for batch, name the file, and click OK.For gathering all cell heights, click on the Detailed tab. Select specific values, and then Position Z from the dropdown menus.
Click the Save icon to export all Z positions and classifications into a XLS file. To begin, pour PBS into a Petri dish under the laminar hood and pipette 20-microliter droplets of the cell media solution onto the inverted lid of the Petri dish. Place the lid back onto the dish and incubate the plate to obtain hanging droplet-cultured 3D spheroids.
To set up the PLOSMA, use a positive displacement pipette to aseptically add 15 microliters of the gel to the wells of a clear 96-well plate. Using a plate spinning rotor centrifuge attachment, spin the plate at 1, 000g for 10 seconds to flatten the gel. Flip the plate 180 degrees and spin again to ensure even gel distribution.
Then, apply focused light at 365 nanometers for 30 seconds to anneal the scaffold and photo crosslink the gel from the top. Aseptically move the Petri dish of hanging droplets into the tissue culture hood and invert the lid. Using a 20-microliter pipette, slowly aspirate a droplet until the spheroid enters the pipette tip and eject the droplet onto the scaffold in the center of the well.
Incubate the well plate at 37 degrees Celsius for two hours to allow spheroids to attach to the scaffold. After incubation, pipette an additional 15 microliters of gel on top of each spheroid. Centrifuge the plate at 300g for 15 seconds in each direction to ensure even gel distribution.
Anneal the top layer of gel for using ultraviolet light as demonstrated earlier. Place the plate under a confocal microscope and image the spheroids after selecting the lowest and the highest Z-planes. After importing the images to the Imaris Arena Software, click the dropdown menu for Channel 1 and select Background Subtraction.
Press OK at the bottom of the panel. In 3D view, press Auto Adjust All Channels in the Display Adjustment popup window and make corrections as needed. In the smaller toolbar, click the Add New Reference Frame icon to create a tab labeled Reference Frame 1.
Move the origin to the center of the spheroid in all three planes. In the same toolbar, click the icon with orange spheres to add new spots, creating a tab named Spots 1, and press the blue arrow button to proceed. For thresholding, adjust the intensity histogram to segment only the brightest parts.
Use the slicer to navigate through the image stack for accuracy. Press the blue Next arrow three times. Uncheck Render on Slicer or click the yellow square icon on the setup panel.
Click on the Statistics tab. In the dropdown menus, select specific values and distance from origin reference frame. Click the Save icon.
To save all changes and analyses, click the Save icon in the main toolbar. This method was utilized to evaluate cellular infiltration at a tissue interface. The median position in the Z-axis of migrating cells increased significantly from zero to 24 hours, indicating notable vertical cell movement.
The fold change in cell migration along the Z-axis doubled after 24 hours. Cell seeded using the PLOSMA method demonstrated significant spreading and migration from the spheroid core after 24 hours. Three-dimensional renderings showed extensive cell spreading in the PLOSMA scaffold after 24 hours, with visible projections extending outward.
The processed 3D images using the Spots Function revealed dispersed cell positions in the scaffold, indicating widespread migration. Cells traveled an average distance of over 200 micrometers with consistent results across samples. The Z-height fold change of the cells in the PLOSMA scaffold was approximately 4, indicating substantial upward movement.
