Proof of Concept

Wet Lab

Our proof of concept involved synthesizing the hydrogel based bio-ink necessary for our proposed 3D bioprinter. The bio-ink must be compatible with the conditions of the 3D printer and, once placed on the canvas, maintain its quality and intensity. Creating reliable bio-ink would encourage artists to consider bioart as a viable and sustainable medium for their art.

Chromoproteins:

Optimizing the chromoproteins which will be used to produce the color in our bio-ink was an essential initial step. Thus, our first goal was developing bacteria with the ability to produce effective chromoproteins. We successfully transformed E. Coli cells with the plasmid containing BBa_K1033926 (part 9F from Kit Plate 6 of the iGEM 2021 DNA distribution kit), which encodes for the asPink chromoprotein. To determine whether the chromoprotein plasmid transformation was successful, we simply identified colored colonies.

However, it is possible that contamination could have produced the pink colored E. Coli. To ensure that the chromoproteins are what are inducing the color, we created liquid cultures of these cells and lysed them. After lysing multiple colonies of cells and getting a pink liquid solution for all of them characteristic of pure pink chromoproteins dissolved in a clear solvent, we were able to prove that the color production was caused by a successful transformation of the asPink plasmid.

Hydrogel:

Our end goal involved synthesizing a hydrogel-based bio-ink that will house chromoproteins. To achieve this, we attempted to construct a plasmid using Gibson Assembly consisting of chromoproteins and hydrogel monomers, which would be transformed into E. coli. If the plasmid was successfully assembled and transformed into the E. Coli, the monomers would polymerize to create a gel that would anchor our chromoproteins, which would serve as our bio-ink.
However, the Gibson Assemblies we ran were largely unsuccessful. Success here would be creating bacteria that, when placed in liquid culture with an appropriate concentration of IPTG, demonstrated a visible color change consistent with the chromoprotein part the plasmid contained. Since the IPTG did not induce color in our liquid cultures, we know that either the Gibson Assembly or the transformation of the Gibson Assembly plasmid failed.
For troubleshooting, we will try putting the pure pink chromoproteins we isolated from the lysis into the hydrogel that was created by PD manually. Additionally, we will continue to run Gibson Assemblies before the competition to create the chromoprotein and csg monomer plasmids and test our results.

Product Development

Our proof of concept involves showing that our 3D bioprinter functions as a form of bioart, as well as showing our handheld bioprinter is an accessible and easy to use variation. By performing extrusion tests and confined compression tests, we can further confirm that our hydrogels have the properties that 3D printers require.

3D Bioprinter:

For our 3D bioprinter, we showed that our design can accurately print the hydrogels into the art sent in by bio artists.

3D Handheld Bioprinter:

Through creating our Handheld Bioprinter, we were able to show that our project can be easily accessible and replicated for all artists to use. Through designing this mini bioprinter, artists will have no trouble using it as a substitute for pencils, painting, and more. We tested factors such as design, extrusion, and accuracy.

Testing Protocol

We designed the handheld bioprinter based on the comfortability and ease of use. This was tested based on how comfortable it was to hold the bioprinter and by printing an entire syringe of hydrogel. Based on our extrusion tests, the bioprinter could be held comfortably and printed successfully.

Testing was conducted using gelatin hydrogel. We tested extrusion by determining if it could extrude the hydrogel smoothly and at a consistent rate throughout the nozzle. Speed could be adjusted through the code by changing the rate of revolution of the stepper motor. Tested at the fastest setting of 15 rpm, extrusion rate was 0.882 mm/s. However, this also revealed inconsistencies in printing. To fix this, we added a 1 mml syringe tip and tested. Testing failed due to presure buildup and we concluded it was more effective to maintain the 3 mm syringe tips. Extrusion can be seen here. Accuracy and precision of the bioprinter was tested by printing sketches of sample bioart and comparing expected results to printed products. From this, resolution, precision, and accuracy could be determined through user testing seen in Figure 11.

In the above video, one can see that in the middle of the printing process, the hydrogel pauses for a second or two before continuing to print. This is due to the fact that when filling the syringe, there were air bubbles that did not allow for further movement of the gel. All in all, the smiley face drawing showed us that our resolution and accuracy can improve with further iterations of the handheld device, in which we can shrink the syringe size for precision and use a different type of hydrogel that does not contain gelatin.

Hydrogel Testing

Making process and result

For the preparation of hydrogels, a novel method was proposed that uses water present in swollen hydrogels as a porogen for shape templates. Crosslinking gelatin with glutaraldehyde in an aqueous solution, followed by rinsing and washing, was used to create biodegradable hydrogels. [2] The aim of our work was to produce hydrogels with various properties. First, we measured out water, about 10mL, and heated it to boiling. After noticing some steam escaping while the water was boiled, we used tin foil to cover the boiling water to reduce water loss. After we measured out the gelatin, in order to create a 3% weight gelatin solution, we dissolved the gelatin in the hot water. Next, a 2 wt% glutaraldehyde solution was added to the 3 wt% gelatin solution in the ratio we were making and stirred briefly. After mixing both solutions, we quickly poured the solution into the mold, and let them stand at room temperature for 12 hours to crosslink. After the ready-made hydrogels were crosslinked, we moved them to a 4-degree celsius fridge for storage and further solidification.

We first experimented with GA:Gelatin ratio 1:10 and 1:5. The result turned out to be that 1:10 ratio hydrogels are hard gel and 1:5 hydrogels are liquidy, and will move if we tilt the Petri dish. Therefore, we decided also to explore the ratio between 1:5 to 1:10 and ratios above 1:10. We made more hydrogels with ratios 1:8,1:9, 1:10, and 1:11. After 12 hours of freezing, they all appear to be fully cross-linked gels. The results are attached here.

Properties Testing

In response to specific physical and chemical stimuli, hydrogels go through a large volume phase shift, also known as a gel-sol phase transition. Temperature, electric and magnetic fields, solvent composition, light intensity, and pressure are examples of physical stimuli, whereas pH, ions, and particular chemical compositions are examples of chemical or biochemical stimuli. The hydrogels are able to revert to their initial configuration following a reaction as soon as the trigger is removed since, in the majority of situations, such conformational shifts are reversible. [3] So we are the first to measure the density and mechanical properties of hydrogels. The density of hydrogels can be calculated by the formula: p=m/v. Here’s a form attached to show different densities of hydrogels.

ratio of GA:gelatin	1:8	1:9	1:10	1:11
Density	1.068667	1.0593	1.198	1.5145

With the help of the Cornell BME department, in particular Dr. Lawrence Bonassar and Leigh Slyker who provided the materials and methods, we were able to perform confined compression tests on the hydrogels. A material's ability to bear axial compressive stresses without expanding perpendicular to the force is measured by confined compression testing. Usually, a closed cylindrical chamber with a sample that entirely fills the chamber volume is used for this test. The sample being tested is put under stress using a piston. Since the liquid phase cannot be compressed, the material must be deformed by at least one wall of the confined compression chamber being porous. The stiffness of the material at equilibrium after fluid flow has halted corresponds to the aggregate modulus (Ha) [1]. Thus, during the confined compression testing that we performed, we used various ratios of our glutaraldehyde to gelatin hydrogels and collected data on how the hydrogels responded to compression. The confined compression test gave us time lapse and displacement on hydrogel after experiencing a certain force.
Getting to know hydrogel properties, we performed various testings. First, we did a confined compression test with premade hydrogels. From the test, we generated a Load vs. Time graph from the data.

• Step 1: Calculate stress and strain using the following equations ⇒ \(Strain = \frac{Change\; in \;Length(dl)}{Original\; Length(l)}\)
• Step 2: Plot the obtained stress and strain data and use the regression equation to obtain Young's modulus. Alternatively, we could simply follow with the following formulas ⇒ \(Stress = \frac{Load}{A}\); \(Strain = \frac{Disp}{L}\).

After conducting the experiment four times, we have chosen the most accurate representation for the 1:10 ratio hydrogel and plotted the load versus time graph.

By analyzing the graph above, we calculated hardness (\(H\)) according to the area under the curve and Elastic modulus (\(E\)) based on the slope of the curve. Modulus is equal to stress over strains.
Here’s the form of 1:10 hydrogels and their Young’s modulus and toughness.

properties/date	0725_035055 1:10	0725_040328 1:10	0725_0402040 1:10	0727_040203 1:10
Modulus	2.2589 Mpa	3.9 Mpa	58.452 Mpa	1.9756 Mpa
Toughness	3517 pa	5294 pa	83787.652 pa	28.7135 pa

From the data we acquired, hydrogels usually have modulus between 0.59 to 2 Mpa. Our 1:10 ratio hydrogel is slightly over the range, which indicates that our hydrogel is relatively stronger to deform. By comparing the data to extrusion testing, we will be able to see which ratio hydrogel will be better in the extrusion test and what properties we will be expecting. Except for the 0727 test sample, all other samples show great toughness and to some sense, this guarantees the continuity of extrusion and makes the hydrogel hard to break.

Cyclodextrin (CD) Nanofiber Incorporation

After testing gelatin and glutaraldehyde hydrogel, we attempted to enhance the mechanical properties of the gel by combining CD nanofiber into it, which have also been used for VOC-uptake applications [2]. Professor Tamer Uyar from Cornell Human Ecology Department kindly offered us CD nanofiber from his lab to experiment on.

According to Professor Uyar’s instructions and previous sources [3] we first cut the nanofiber into very small pieces and soaked them in warm water to break them down faster. After a day sitting on the bench, we used a magnetic stirrer to stir them for some time until they were really wet and were floating all over. We performed sonicating with pulse on 3 seconds, pulse off 4 seconds and repeated for 20 mins to further disperse them in water. The process pictures are shown below. Our nanofiber is not breaking down at a speed that we aspired to, and currently we are trying to increase the efficiency. Here is the CD Nanofiber Procedure for our experiment.

If we successfully made the CD nanofiber hydrogel, we would be testing its properties and perform VOC uptake testing as well.

Sources

[1] Biomomentum. (2022). CONFINED COMPRESSION. Biomomentum. https://www.biomomentum.com/mechanical-test-configurations/confined-compression/

[2] Celebioglu, A., Sen, H. S., Durgun, E., & Uyar, T. (2016). Molecular entrapment of volatile organic compounds (VOCs) by electrospun cyclodextrin nanofibers. Chemosphere, 144, 736-744.

[3] Yu Huang, Xiufang Li, Zhentan Lu, Huan Zhang, Jiangxi Huang, Kun Yana and Dong Wang a. (2020). Nanofiber-reinforced bulk hydrogel: preparation and structural, mechanical, and biological properties. ROYAL SOCIETY OF CHEMISTRY. https://pubs.rsc.org/en/content/articlelanding/2020/tb/d0tb01948h#!divAbstract