The overall goal of this procedure is to study the migration characteristics of cells in 3D physiologically relevant environments. This is accomplished by first seeding cells in an inner collagen gel that is then enclosed in a cell-free outer collagen gel with the desired matrix properties. Next, the cells are allowed to invade and migrate in the outer gel during which they can be monitored and quantitatively analyzed.
Ultimately, this concentric gel assay is used to show how cell migration behavior is affected by the biophysical properties of the extracellular matrix. The main advantage of this technique over existing methods like the transfer assay or the scratch assay, is that the migration of the cell happens in a physiological relevant treaty environment whose structural and biophysical properties are very easy to control. Visual demonstration of this method is quite essential because the formation of the concentrate inner router gel system is quite delicate.
A proper gel interface is very important for the success of this experiment, which can sometimes be a bit challenging to achieve. Begin with a culture of MD MBA 2 31 cells ready to harvest from T 25 flasks at 80%co fluency. First trypsin is the cells using a milliliter of solution per flask.
Then collect the cells as a pellet using 200 Gs of force for four minutes, discard the snat and resuspend the cells in five milliliters of culture.Media. Now measure the cell density using a hemo cytometer and determine the volume needed for 100 million cells per milliliter. Then spin down the cells at 200 G for four minutes.
Remove the supernatant and thoroughly resuspend the pellet into the required volume of serum free culture medium. This suspension is at 10 times the final seating concentration. To begin gather the required stock solutions in an ice bucket, they include 10 XPBS milq water, and 0.1 molar sodium hydroxide.
Also chill down several micro centrifuge tubes under a hood, maintaining sterile technique, aliquot out the amount of collagen desired for 50 microliters of 2.4 milligrams per milliliter inner gel solution. Then slowly add five microliters of 10 XPBS to the measured out collagen. Swirl the tube as the PBS is added, but avoid making bubbles.
Next, adjust the pH of the mixture to 7.4 with the base solution. Usually five microliters will do the job. A troubleshooting guide is provided in the text protocol.
Lastly, bring the volume up to 45 microliters with serum free culture medium leaving room for the 10 x cell suspension. To begin, add five microliters of 10 x cell suspension to the collagen solution and resuspend the cells thoroughly in the solution without forming air bubbles. Now to a prewarm to glass bottom plate, add 20 microliters of the suspension to the center.
Well forming a dome shaped droplet if a bubble forms quickly rupture it or suck it up without making a mess. Casting the gel the right way can be very tricky, especially with very viscous solutions like collagen. Try to avoid producing bubbles as much as possible and don't swirl the solution once it is ready to polymerize, because swelling might result in artificial fiber alignment.
Now return the dish to the incubator for 45 minutes to let the inner layer polymerize 15 minutes before the incubation ends. Prepare 200 microliters of outer collagen gel solution on ice. First, aliquot out the required volume of stock collagen.
Second, slowly add 20 microliters of 10 XPBS with gentle swirling. Then adjust the pH to 7.4 as before, and bring the solution up to 200 microliters with Milli Q water. Now remove the dish from the incubator and gently add 180 microliters of the outer gel solution on top of the inner gel dome.
This should completely cover the inner solution and fill the well. Do not stir the solution and avoid touching the inner gel solution. If a bubble forms while ejecting the outer solution, be quick to rupture it or suck back into the pipette.
Another important step is to make sure a proper gel interface is obtained. The key to this step is to add the outer gel solution. When the inner gel is yet to certify.
The geling kinetics can vary with polymerization condition and it is essential to optimize this condition beforehand To let the outer gel polymerize return the dish to the incubator for 45 minutes. After 45 minutes, the gel should be fairly solid, but can still be dislodged carefully add two milliliters of warm medium to fully submerge the gel. This completes the concentric gel culture provided fresh medium every two to three days.
Within days, cells will begin migrating into the outer matrix to observe their migration, stain the cells with a fluorescent tracker. Replace the two milliliters of medium with fresh medium containing five microliters of dye and incubate for 30 minutes after the incubation period, wash the culture using one milliliter of PB S3 times for two to three minutes per wash. After the washes, refill the dish with normal media.
Then set the dish up on an environmentally controlled microscope. Now use a volume of use setting to scan the outer gel regions next to the gel interface. Typically scan in 647 micron square views that are 100 microns thick, using five micron intervals on the ZS stack.
Then scan the intermediate regions and finally scan the outskirts of the gel. Exclude regions within 50 microns of the bottom, top, or side surfaces to avoid cells that might be suffering from an edge effect. To analyze the movement of the cells from the collected data first open the image files in data analysis software such as imas.
Begin by selecting the command surpass and then the command surface. This pulls up the option to track surfaces. Over time, select the region of interest within the dataset, meaning the Cartesian coordinates and the timeframe.
Next, subtract out the background from the region of interest. To avoid losing a cell to the background elimination, select the largest sphere that fits into the object. Now, split the connected objects by selecting the seed point diameter and proceed to classify all the seed points.
The next step is to classify the surface Now manually. Group seed points that point to the same object. Do this between all the time points in the region of interest.
Finally, track the objects when tracking is completed, classify the object tracks, and then output the quantitative data set to analyze in using a spreadsheet. After a few days of concentric gel cultures, cells breached the inner outer gel interface and started to invade the outer gel. The cell population spread outwards in a predominantly radial direction.
The movement of cells in the outer gel of various collagen densities was compared. Imaging was performed over an eight hour period, and about 200 cell trajectories were calculated from each sample. Cells in the thinnest matrix moved the least distance.
This calculation took into account all of the cells, not just the cells migrating radially outward. In all cases, the total distance covered was greater than the net displacement. As a expected persistence was calculated as a ratio between displacement and distance.
The persistence value of 0.4 suggests that these cells had an intrinsically weak directional migration. Mean speed calculations showed that the collagen density did not affect the cells speed. It is possible that matrix pore size and collagen density, which are inversely related, had the opposite effect on migration speed and thus offset each other.
After watching this video, you should have a good understanding of how to build a concentric gel system and how to study this migration of cells in 3D environment. Following this procedure, you can do a lot of other things like cell extraction, microarray analysis, where you can try to answer additional questions like how much certain proteins are expressed during different time points in the migration.I.
