Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

The overall goal of this protocol is to engineer three dimensional collagen scaffolds that can resemble the architectural features, composition, and mechanical properties of in-vivo tissue. This method can help answer key questions in the tissue engineering field, such as ECM cell interactions, the influence of architectural features in the creation of engineered tissues, and the development of tissue replacements, among others. The main advantage of this technique is that it provides a viable procedure to recapitulate the microgeometry of interior flow channels and paths with high fidelity in a biodegradable material.

Demonstrating the procedure will be Veronica Rodriguez-Rivera, a post-doctoral associate in my laboratory. The materials used to prepare the BSA rubber must be stored and kept cold until they are ready to use so that the BSA rubber does not prematurely set. Sterility is also very important to this procedure.

First, sterilize the dispenser. In a non-cell culture hood, lay out the O rings, the syringe, its cap, the mix tip, and the four-to-one dispenser. UV sterilize these materials for 30 minutes.

First, screw the two stainless steel mold pieces together and attach the Luer lock. Before assembling the dispenser, spray pipettes and reagents prior to introducing them to the hood with 70%ethanol. First attach the tip cap to the solution holder.

Next, load the 30%BSA solution into the larger chamber without contaminating the other chamber. Leave enough room to attach the O ring. Then, load the smaller dispenser chamber with three percent glutaraldehyde and attach another O ring.

Now, attach the syringe to the dispenser. Then tilt the assembly so the cap is on top, and replace the cap with a mixing tip. Then gently tap the sides to allow the air bubbles to travel upwards.

Next, place the dispenser inside of an autoclaved bag to contain the exhaust solution. In the bag, with the dispenser upright, dispense a small amount of solution to clear all the air from the syringe. Removing the air is very important.

Then, quickly, attach the stainless steel Y mold's Luer lock to the syringe tip. Now, with the mold in your non-dominant hand, alternate ejecting solution into the left and right exhausts of the outflow channels. Press the exhausts, thus filling the internal voids with solution.

Then, position the mold horizontally and inject it with more solution until half the dispenser volume has been used. The mold should now be placed into a dish, wrapped in parafilm, and stored at four degrees Celsius overnight. This procedure is scaled to four grams of acidified collagen.

Keep it chilled until it is used. After the collagen has been weighted, proceed to sterilize the solution by gamma radiation. In preparation, make fresh 0.2 normal HEPES solution in water.

Then transfer the contents to a 50 milliliter conical tube. Adjust the pH to nine using concentrated sodium hydroxide. Be sure to UV-sterilize the hood and autoclave the forceps, spatula, and scalpel.

In the hood, mix 1.5 milliliters of the HEPES solution with 1.5 milliliters of 10x MEM. Store this mixture on ice. Also, chill a 12 well plate and a 20 milliliter syringe in the freezer for 10 minutes.

For added sterility, spray the tubes and plates involved with 70%ethanol prior to placing them in the hood. Spray the mold plate, and place it in the hood. Then, remove the BSA rubber molds with forceps.

Then, cut the exhaust channels off the mold using a scalpel. Next, briefly vortex the HEPES-MEM mixture and add one milliliter to the collagen. Then, thoroughly mix the solution with a sterile spatula.

Follow with a brief vortex, and quickly transfer the solution to the cold, 20 milliliter syringe. Now, while securing the BSA rubber inside of the well, dispense half the collagen hydrogel solution into the well bottom. Next, using tweezers, position the mold so that the rubber inflow and outflow ends are touching the walls of the well.

Then, cover the mold with collagen hydrogel. Now, seal the well with parafilm, and load the plate into a 37 degree Celsius incubator. Allow one hour for the collagen to polymerize.

During this time, keep the UV light in the hood on. Also, set the UV crosslinking apparatus to deliver 630, 000 microjoules per square centimeter. Now, spray down your gloved hands with 70%ethanol, and load the plate with the collagen covered molds into the crosslinker, and start the irradiation process.

After the irradiation, spray your gloves again, and unload the plate from the crosslinker. Then, using a sterile spatula, remove the gel from the well flip it over, and crosslink the bottom of the hydrogel. After the final crosslinking cycle, proceed with the enzyme digestion.

To remove the BSA rubber without disturbing the collagen scaffold, use sterile 0.25%trypsin solution at pH 7.8 and press it through a 0.20 micron filter. Transfer enough filtered trypsin solution to a new 50 milliliter conical tube. Then, transfer the collagen hydrogel to the conical tube that contains the enzyme solution.

Make sure that the hydrogel is completely covered with the solution. Seal the tube with parafilm, and vortex it gently for about a minute. Then, incubate the tube at 30 degrees Celsius and let the digestion go for 15 to 24 hours.

For the first four hours, remove the tube every 15 to 30 minutes and gently vortex it to help complete the BSA rubber digestion. The next day, the digestion is considered complete when no rubber floats in the solution or there are no broken down pieces of the rubber in the hydrogel. Now, replace the trypsin solution with sterile Mosconas solution, and let the tube shake at four degrees Celsius for 30 minutes.

Later, aspirate the Mosconas solution and repeat this step two more times to fully clean the collagen scaffold. An extensive number of tests were used to optimize the use of the biorubber and described in detail in the text protocol. Using the optimized protocol and three solid mold pieces, collagen hydrogels were fabricated and examined in detail.

The Y mold needed was crafted on a Microlution machine. The Y mold was injected with 30%BSA and three percent glutaraldehyde, which reacted overnight at four degrees Celsius. Next, the BSA rubber was casted with the collagen and heated to 37 degrees Celsius for an hour.

The rubber was enzyme digested for 15 hours, which weakened it to the point of falling off without affecting the geometry of the collagen hydrogel. The four millimeter inflow channel cast by the mold was measured with calipers to be precisely four millimeters in the collagen hydrogel. Testing the stability of the mold, it was found that dimensions as small as 300 microns could be reliably engineered.

In addition, the scaffolds were tested for residual glutaraldehyde after the Mosconas washes, and no residue was found. After watching this video, you should have a good understanding of how to fabricate 3D collagen scaffolds with the potential to mimic in-vivo geometries. Following this procedure, other methods that complement this technique, such as creating 3D models of in-vivo tissues and fabricating molds with specific architectural features can be performed to answer additional questions.

After its development, this technique paved the way for researchers in the field of tissue engineering to explore the interaction between internal architectural features and matrix components with cells in in-vitro models.