Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

The overall goal of this procedure is to print a reverse mold to create patterned two component hydrogels for 3D cell culture. This is accomplished by first printing a reverse mold from a thermo reversible polymer, which is a liquid at four degrees Celsius, but a soft gel at room temperature. This reverse mold can later be eluded.

The second step is to fill the reverse mold with a biopolymer of choice. Here we used aros. The biopolymer can be cross-linked either by enzymatic, UV, or temperature cross-linking.

Next, the reverse mold is El alluded by submersing the mold in cold water. After this step, the resulting AROS mold is ready for further processing. The final step is to fill the voids with a second biopolymer, which could contain cells and can be cross-linked by means of enzymes, UV light, or temperature.

Ultimately, we have established a reproducible method to create spatially organized to D cell culture scaffolds. We first had the idea for this method when a colleague suggested we could take advantage of the thermo responsive properties of Oxr by using it as a sacrificial mold. Demonstrating the procedure will be Misha Muah, a graduate student from my laboratory.

To begin add 60 milliliters of ice cold PBS solution into a glass bottle and stir vigorously using a magnetic stir. Then weigh out 24.5 grams of Paul ER and add it in small amounts to the cold PBS. Wait until the Paul Oxr has partially dissolved in the PBS before adding more.

Continue to stir the solution until all Paul Oxr has dissolved. Next, add additional ice cold PBS until a final volume of 100 milliliters is reached, producing a final concentration of 24.5%weight per volume. Then stir the mixture until it is well mixed.

Stop stirring the solution and let it rest at four degrees Celsius overnight until bubbles and foam in the solution have disappeared. Bubbles that are trapped within the gel are transferred to the printer cartridge and will lead to defects in the printed sacrificial molds. Sterile Filter the solution directly into the printing cartridge using a pre-cool filter to remove any unwanted particles that could clog the needle.

Store the cartridge at four degrees Celsius until 30 minutes before the experiment. Prepare the solenoid valve and needle for printing by placing them in separate 1.5 milliliter micro centrifuge tubes filled with ultrapure water and syndicating them for 30 minutes. Then rinse the cleaned valves with ethanol and dry them with a nitrogen gun.

Next, install the clean solenoid valve and needle in the printer. Also, install an empty clean print cartridge to test the system. Apply three bar pressure to the system and blow out any residual liquids from the installed valve and needle with compressed air.

For small needle diameters. Install a filter at the exit of the compressed air to avoid entry of small particles that could clog the needle. Then turn the pressure off.

When the pressure has dropped to zero, install the cartridge preloaded with the poly oxr. The cartridge should be taken out of the refrigerator approximately 30 minutes before mounting the cartridge so the still liquid poly oxr can reach room temperature and gel before printing. Check if the poly oxr is jelled by flipping the cartridge.

Next, apply three bar of pressure to the system and dispense Paul Emer until it reaches the needle tip and is extruded in a continuous strand. Using the bio CAD software, draw a single line about the same length as the structure that you intend to print in the printer software settings menu. Set the solenoid valve to a high frequency of 50 hertz in the parameter section and hand tune the high pressure setting to three bar.

Next, set a glass microscope, slide in the printer and secure it into place by turning on the vacuum. Then print one layer of a single line with a stage speed of 300 millimeters per minute. Obtain the desired line width by reducing the pressure by hand and the frequency settings in the software for each new line, make sure the line remains continuous.

If the line drawn is broken, the lower limit has been reached. Once a continuous line with the desired width is achieved, determine the optimal stage speed and layer thickness by checking for defects in the printed constructs. In order to fine tune the layer thickness, the pressure and frequency may also need to be slightly readjusted.

In order to test out the layer thickness setting, print several layers on top of each other and watch to make sure the needle is in the right position above the previous layer. Adjust the layer thickness so that each layer is printed directly on top of the previous one. To optimize the stage speed begin by decreasing the stage speed from 300 millimeters per minute stepwise so that extruded layers start and end at the same positions as the previous ones.

If the stage moves too fast, a wave-like pattern will appear on the top of the layer. For printing pillar structures, draw a single point instead of a line in the CAD software. Then fine tune the pressure and frequency to regulate layer thickness and pillar diameter and the step time of the print head to maximize printing speed.

Once optimized, save the parameters for later use. First, print the inner structure such as this pillar array on a glass microscope slide and let it dry. Overnight drying reduces the size and thickness of the structures and provides better adhesion between the structure and the substrate.

The next day, draw a structure that consists of an outer wall surrounding the structures you intend to have eluded away and filled. The polymer wall should be printed at least 3.5 millimeters away from the inner structures due to the dimensions of the needle. Next, prepare a mixture of 1%aros in deionized water by heating it in the microwave once fully dissolved.

Cool the agro solution to between 35 and 45 degrees Celsius. Then fill the sacrificial mold with the agro solution using a pipette. This should be done slowly to avoid destruction of the structure inside the wall.

Once filled, place the mold at four degrees Celsius to solidify the gel. After about 10 minutes, remove the gel from the fridge and place it into an ice bath for 10 minutes to elute the polymer structure. Then remove the structure carefully and remove residual water by blotting it with a paper tissue.

Press the structure carefully onto a new glass microscope slide to create a good seal preventing leakage of the third hydrogel. Next, prepare a 1%solution of alginate methacrylate in 0.15 molar sodium chloride. Vortex the solution to dissolve the alginate methacrylate.

Then add 2.5%volume to volume. Alexa, 4 88 conjugated fibrinogen, and 0.05%weight per volume. Lithium phenyl 2 4 6 trimethyl benzoyl phosphate to obtain a third hydrogel that can be easily visualized via fluorescence.

Place this solution into a 0.3 milliliter syringe equipped with a 30 gauge needle. Then use the syringe to fill the voids left by the eluded polymer. Be sure to completely fill each column by ejecting the polymer as the needle is drawn from each column.

Finally, photo polymerize the injected polymer with a high intensity UV lamp for five minutes, and then image the construct using an epi fluorescence, or confocal microscope. Shown here is a 3D Zack reconstruction of a construct similar to the one that was just fabricated, the fluorescently labeled alginate used to backfill. The pillar shapes appears as green columns and the outer part of the construct is transparent.

The techniques shown in this video are widely adaptable to many different 3D arrangements, such as the concentric circles shown here. Resolution is dependent on the needle diameter. The 150 micron diameter needle used for this design can produce strands between 120 and 200 microns in diameter.

After watching this video, you should have a good understanding of the steps involved in printing oxr. We have demonstrated the use of oxr as a sacrificial mold to create pattern hydrogels, but the oxr can also be UV stabilized if a manufacture related version of the polymer is used.