Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

This method allows us to engineer 3D collagen hydrogels with controlled levels of fiber alignment to model environments found in healthy and diseased tissue in the body. This technique provides a simple microfluidic approach to engineer aligned collagen hydrogels, introduce cells, and quantify how cells behave in topographically defined 3D environments. Demonstrating the procedure will be Neil Joshi, Mehran Mansouri, Ann Byerly, and Justin Vidas for my laboratory.

To begin, mount a 250-micrometer thick PDMS sheet onto a plastic carrier and razor cut the microfluidic design using a craft cutter at a blade depth of 0.5 millimeters, speed of one centimeter per second, and high force. Using an ultrasonic bath, clean the microfluidic channel cutouts for five minutes. Rinse the sonicated channels in deionized water and dry them on a hot plate at 100 degrees Celsius for five minutes.

Store the channels in a clean, covered Petri dish until use. To fabricate the PDMS cover layer and functionalize the surface for future modification, prepare a 2%aminopropyl triethoxysilane solution in a glass beaker by adding one milliliter of aminopropyl triethoxysilane to 49 milliliters of acetone. Next, dilute a 25%glutaraldehyde solution to 5%in deionized water.

Make two milliliters of solution for each 24 millimeters by 50 millimeters cover slip. Clean the cover slips in a bath sonicator for five minutes using IPA. Rinse off the IPA from the cover slips using deionized water.

A smooth film of water on the cover slip indicates that the IPA is thoroughly rinsed. Dry the cover slips on a hot plate for five minutes at 100 degrees Celsius. Place the dried cover slips into clean Petri dishes, ensuring they do not overlap.

Using a corona discharge wand, expose the cover slips to a corona discharge for one minute each. Remove the cover slips within five minutes of corona exposure and immerse each cover slip into the aminopropyl triethoxysilane solution for 10 seconds, ensuring the cover slip is submerged Then, remove the cover slips from the aminopropyl triethoxysilane solution, immerse them into acetone for 10 seconds, and dry them with compressed air. Place the dry cover slip back into the Petri dish, the treated side facing up.

Pipette one milliliter of glutaraldehyde solution onto the surface of each cover slip. Cover as much surface as possible without allowing the solution to spill over the edge of the cover slip. Let the cover slips sit in contact with the solution for 30 minutes and then rinse with deionized water for 20 seconds.

Dry the cover slips using compressed air and place them back in the Petri dishes, the plasma-treated side up For laser cutting the modular magnetic base, cut out the designs from the PMMA layer using appropriate laser settings, such as the number of passes and power. The laser settings should be adjusted so the magnets can be press-fitted in the PMMA layer. Wash the laser-cut part using soap and water to remove debris from the laser cutting process.

Do not use solvents, as they may propagate micro-cracks in the laser-cut edges. To assemble the platform, push magnets by hand into the laser-cut base. The thickness of the magnets must be less than the thickness of the PMMA base to ensure the magnets are flush with the surface of the base.

Peel off the backing from the pressure-sensitive adhesive, or PSA sheet, and attach the base to a glutaraldehyde-treated cover slip with the functionalized side facing up. Gently place the PDMS channel cutout into the cavity defined by the frame. Press down with wide-tip tweezers to remove air bubbles and ensure conformal contact.

Place the bovine serum albumin, or BSA-treated channel cover on top of the channel cutout with the BSA side facing down. Ensure the fluid inlet and outlet ports are aligned with the channel. The device is ready for the collagen I injection.

Place a syringe pump, a chilled sterile syringe, chilled neutralized collagen I solution, and a sterile 20-gauge 90 degrees angle-tip luer lock needle into a biosafety cabinet. Load the collagen I solution into the syringe, avoiding bubbles. Attach the needle tip to the syringe, load the syringe into the syringe pump, the needle facing down, and prime the needle with collagen I solution.

Set the syringe pump to the required flow rate between 50 to 2, 000 microliters per minute. Place prepared PDMS channels on a lab jack and level with the needle. Insert the needle into the inlet port of the PDMS channel.

Inject the channel until a 30-microliter drop of collagen I collects on the outlet side. Lower the lab jack and gently separate the needle from the newly filled channel. Repeat until all channels have been filled with collagen I solution.

Load the filled channels into the Petri dishes alongside a clean, lint-free wipe saturated with deionized water to prevent dehydration of the newly formed collagen I gel. Cover the Petri dish and place the loaded channels in the incubator for two hours before the peel-off step. For peel-off and media equilibration, start by lifting off the PDMS cover using tweezers to expose the polymerized collagen I gel.

Add 650 microliters of endothelial growth media to the well. Alternatively, attach another module to introduce an additional collagen layer or provide media perfusion capabilities. Leave the devices in the incubator for a minimum of four hours to equilibrate the gel and media.

Replace the media before seeding with cells. Functionalizing the glass cover slip enabled channel liftoff and functionalizing the PDMS cover with BSA prevented collagen I attachment. The collagen I fiber alignment remained unaffected after lifting off the cover.

Images of the HUVECs cultured on the aligned segment of the matrix showed increased actin fiber alignment compared to the HUVECs on the segment with random fibers. Our approach allows us to model the tumor microenvironment and quantify how different cell types respond. We can also introduce flow channels to support long-term experiments.