Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The overall goal of this procedure is to create a planer gradient diffusion chamber to investigate chemo taxes in a 3D collagen matrix. This is accomplished by first fabricating a 3D silicone mold with the use of a live cell imaging chamber. The second step is to make hydrophilic and hydrophobic cover slips and to use them to assemble the 3D silicone mold.

Next, the collagen cell mixture is added to the center compartment and allowed to polymerize. The final step is to remove the hydrophobic cover slips from the mold after incubation and to add plain media to one reservoir and media with chemo attractant to the other reservoir. Ultimately, confocal microscopy is used to track cell chemotaxis over time and visualize cell motion displacement within the collagen matrix.

The main advantage of this technique over existing techniques like the boin chamber and micro athletics, is it allows for a simple setup to investigate migration in 3D space with a simple diffusion gradient, No using a disposable pipette. Measure out 150 microliters of the amino seline A-P-T-M-S and pour it in a 50 milliliter tube. Then add 30 milliliters of 100%ethanol to the tube with a second disposable pipette, and close the lid.

Vortex the solution for two minutes to ensure complete mixing. Then pour the solution in a glass Petri dish and set it aside. Next, pour 15 milliliters of 100%ethyl alcohol in a second Petri dish and place it with the first dish using a new disposable pipette.

Measure out 30 milliliters of deionized water and pour the solution into a 50 milliliter tube. Next, use a new disposable pipette to add 1.875 milliliters of glu aldehyde to the tube and close the lid. Vortex the solution for two minutes, and then pour the mixture into a third Petri dish.

Using forceps, take out 1 22 millimeter round glass cover slip and rinse both sides with 100%ethanol mixture. Using a disposable pipette, place the rinse glass cover slip into the first dish that contains amino seline and allow it to sit in the solution for five minutes using forceps. Take out the glass cover slip and rinse it again with 100%ethanol.

Then drop the rinsed glass cover slip into the dish with glutaraldehyde and set it aside for 30 minutes. After 30 minutes, remove the glass cover slip with forceps and rinse it with deionized water. Place the rinsed cover slip onto a tissue to dry overnight at room temperature.

To prepare hydrophobic cover slips, first add 500 microliters of TTS 100 microliters of acetic acid and 19.4 milliliters of heane in a 50 milliliter tube and vortex it for two minutes. Then pour the solution into a glass Petri dish and use clean forceps to drop one glass cover slip into the prepared solution and leave it there for two minutes. After two minutes, take out the glass cover slip using forceps and rinse it with deionized water using a disposable pipette, place the cover slip onto a tissue and allow it to dry at room temperature overnight.

Store the dried cover slips in a plastic Petri dish until they are needed to begin, assemble the live cell imaging chamber as described in the accompanying text protocol. Next, mix the silicone elastomer solutions according to the manufacturer's instructions to make five milliliters of elastomer. Then use a disposable lab spatula to slowly pour the silicone elastomer solution into the live cell imaging chamber setup so as not to disturb the mold components.

Set the mold aside in a safe location to curate room temperature overnight. The next morning, pull out the mold using forceps and carefully extract the machined aluminum metal cube from the mold. Next, go to the sink and rinse the mold with deionized water, and then place the mold on a paper towel to dry.

Once dry, use a hobby utility knife to cut slits space 2.34 millimeters apart from each longitudinal end inside the silicone mold. Ensure the mold stays in a safe and dry place until needed for the experiment prior to use. Rinse the mold with 90%ethanol and place the mold into a Petri dish filled with deionized water to sit overnight.

The next morning, transfer the mold to a paper towel and allow it to air dry while the mold is drying. Use a high precision diamond scribing tool to cut the square. Hydrophilic cover slips into two rectangles, slightly larger than 3.95 millimeters by 5.99 millimeters.

Then slide each of the cut rectangle cover slips into the slots, cut into the silicone mold, flip the mold upside down and apply vacuum grease along the bottom of the silicone mold using a disposable pipette. Next, flip the mold back right side up and press the bottom of the mold onto the circular hydrophilic glass cover slip. To create a seal, prepare a mixture of media and chemo attractant in a 15 milliliter tube at the desired concentration, pipette five milliliters of the mixture into a new milliliter tube and five milliliters of plain media into a second tube.

Move both tubes into a water bath heated to 37 degrees Celsius. Next pipette 30 microliters of 10 XPBS into a micro centrifuge tube. Add six microliters of a one normal sodium hydroxide solution into the same micro centrifuge tube and vortex for 15 seconds.

Then add 18 microliters of yellow green fluorescent carboxylate, modified microspheres to a micro centrifuge tube and 168.3 microliters of ice cold. Type one rat tail collagen. Vortex the mixture for 30 seconds.

Next, add 77.7 microliters of the desired cell. Type at two times 10 to the six cells per milliliter in media to the micro centrifuge tube and mix. Using a pipette tip for 20 seconds, pipette 300 microliters of the collagen cell mixture into the center well of the prepared mold assembly.

Then place the mold onto a disposable Petri dish and move it into a standard incubator for 20 minutes at 37 degrees Celsius with 5%carbon dioxide After the incubation period, use forceps to remove both of the hydrophobic cover slips by clamping onto the top of each cover slip, and then pulling up and away from the mold. Move the whole system to the microscope and examine the chamber to ensure the sides of the collagen mold are still straight, allowing for a planer diffusion gradient with a system still under the microscope. Add 100 microliters of cell media into the first well.

Next, add 100 microliters of cell media with the desired chemo attractant concentration into the well on the other side of the matrix. Track the cell migration in reference to the fluorescently labeled microspheres as described in the accompanying text protocol. The series of images presented here show the diffusion of rumine moving through a one milligram per milliliter collagen gel.

This example displays the system's true planar diffusion gradient, which can also be expected of any introduced chemo attractants. In the example shown here, the intensity of the rumine signal was measured over time at the locations identified by the red squares. The concentration at each of these locations is shown on this graph as a separate line, which when measured at steady state, produces a linear concentration gradient based on position in the gel.

This relationship remains linear throughout a range of collagen matrix concentrations making this system very useful for cell experiments. In this image, a neutrophil colored red is shown within a collagen matrix scattered with fluorescent green microspheres that are used as position references. The next three images show the directed migration of the neutrophil as it moves up a chemo attractant gradient, which is depicted by the white arrow.

After watching this video, you should have a good understanding of how to create a simple diffusion chamber to investigate cell migration in a 3D collagen matrix.