Before we incorporate any living organisms into the biomaterial, we wanted to optimize our protocol and determine the operable range of each component in the bioink. Learn more about our bioink composition and how it is created in this page!
The composition of our bioink is inspired by Reinhardt et al.’s methodology, which consists of a hydrogel-based bioink capable of extrusion bioprinting ([1]). The stabilizing and biocompatibility properties of the 3wt% alginate and 9wt% methylcellulose formulation has led to higher viscosity, better shear recovery and better printability ([2]). As a result, this was the only bioink to be selected by companies to be used and tested in the International Space Station ([2]). Thus, our bioink consists of:
Sodium Alginate - a commonly used biomaterial for bioprinting in bacterial, mammalian, and plant cells ([3], [4]). Its shear-thinning properties, which makes it highly viscous at low shear rates, and lower in viscosity at high shear rates, is optimal for extrusion-based bioprinting.
Carboxymethylcellulose (CMC) - a biopolymer that is commonly used as thickeners for gel formation, allowing the ink to have a stronger structural and stacking ability ([5]). Compared to its similar derivative, methylcellulose, CMC maintains a more consistent viscosity and is stabler across a wider temperature range ([6]).
Construction Material (Sand) - the sand will serve as the material that will be cemented together by calcium carbonate crystals to form bricks. We tested two compositions of sand:
Sakrete Multi-Purpose Sand (Earth) - affordable and abundantly sourced dry-screened sand used for construction and cement-based mixes
Mars Global High-Fidelity Martian Regolith Simulant (MGS-1) - sourced from Space Resource Technologies, MGS-1 is a collection of terrestrial minerals and oxides to generally replicate the Martian surface based on data collected from the Mars Science Laboratory Curiosity rover ([7]). The tables below contains a list of components in MGS-1. Table 1. Mineralogy and bulk chemistry composition of MGS-1 ([8]).
Component
Wt. %
Oxide
Wt.%
Anorthosite
27.1
Silicon Dioxide
43.90
Glass-rich basalt
22.9
Titanium Oxide
0.46
Bronzite
20.3
Aluminum Oxide
12.84
Olivine
13.7
Ferrous Oxide
10.60
Mg-suflate
4.0
Manganese(II) Oxide
0.11
Ferrihydrite
3.5
Magnesium Oxide
14.81
Hydrated silica
3.0
Calcium Oxide
7.91
Magnetite
1.9
Sodium Oxide
1.49
Anhydrite
1.7
Potassium Oxide
0.29
Fe-carbonate
1.4
Phosphorus Pentoxide
0.17
Hematite
0.5
Loss on Ignition
4.90
In order for alginate to form a gel, it requires divalent cations to ionically bind to the alginate polymer chains, in a process called crosslinking. Calcium ions are the most commonly used divalent cation for crosslinking, and is supplied using a calcium chloride (CaCl2) solution. The concentration often used is 100mM of anhydrous CaCl2 dissolved in deionized water.
Before we incorporate any living organisms into the biomaterial, we wanted to optimize our protocol and determine the operable range of the different components as adapted from Reinhardt et al.’s formulation ([1]). Thus, we asked:
What is the operable range of carboxymethylcellulose and earth sand/Martian regolith in 3wt% alginate that can be crosslinked with a 100mM calcium chloride solution?
Methodology
We followed the protocol outlined below to create our gel formulations:
Prepare a 3wt% alginate solution (in de-ioinized water)
Notes for mixing:
Use a magnetic stir plate that has a hot plate feature
Start by adding water to an appropriately sized beaker and using a magnetic stir bar, create a vortex (ensure that magnetic stirring is high)
Once a vortex is created, gradually add alginate, ensuring that it is mostly dissolved before adding more.
Prepare a 100 mM calcium chloride solution for the baths
Sieve the sand according to the below conditions
Table 1. Weight % of sand (across) and sieve size (down)
30 wt%
40 wt%
50 wt%
60 wt%
70 wt%
850 µm
425 µm
150 µm
Combine 9wt% CMC powder (0.9 g) and x wt% sieved sand according to Table 1 conditions for 10mL of gel
Add the CMC and sand mixture to 10 mL of alginate solution and mix with a spatula
Measure the weight of the gel made, then mix in 100 µL of deioinized water (or bacteria suspension) per g of gel
Fill the paste into the 10 mL syringes
Extrude the gel in various shapes into a petri dish, such as:
in a rectangular shape, layering 3 times (shown in Figure 1)
in a straight line 3 times (shown in Figure 1)
in a solid rectangular shape
Submerge the gel in 100 mM calcium chloride solution and cross link for 10 minutes
Observe whether:
The gel clogs the syringe
Layers are stable and hold on to each other
Figure 1. Scaffolds of a 3-layered hollow rectangle (top) and 3 straight-line extrusions (bottom) crosslinking in 100mM Calcium Chloride solution in a petri dish.
Dr. Nicholas Lin has experience developing bioinks incorporating mycelium. Thus, we approached Dr. Lin and had multiple meetings and training on how to optimize and test bioinks. Dr. Lin advised us on how to calculate components needed based on w/v% and demonstrated how he prepared his bioink composed of carboxymethylcellulose (CMC), cornstarch, and agar powder. He suggested that to optimize the bioink composition, the two main goals are that the gel is able to 1) extrude through nozzle and 2) layers are able to build on each other. This motivated our methodology above in seeing what the maximum weight % of sand and/or particle size is for smooth extrusion.
Dr. Nicholas Lin
Postdoctoral Fellow, Hallam Lab, UBC Vancouver.
Testing with Earth Sand
DBTL 1 - Earth Sand
Before finalizing the formulation of our bioink end product, we first aimed to optimize the ideal bio-ink composition (i.e., structural integrity and stability) before adding our transformed bacteria.
We achieved this by creating a protocol (listed in methodology) that served as the baseline for our future bio-ink formulations. Here we tested for various factors such as 850, 425 and 150 µm particle size and 30-70 weight % sand.
We carried the protocol out with Earth Sand first, noting how easy it was to extrude the sand from the syringe and whether the scaffold can maintain its structure with crosslinking.
We learned what steps of the protocol needed to be optimized for better gel formation and what can be concluded based on particle size and weight % for Earth Sand.
From the initial experiment conducted on 2025.05.26 Earth Sand Alginate Gel Test (850 µm), we found that sand weight % did not play a significant role in ease of extrusion. However, 850 µm was too large of a particle size for extrusion, as it resulted in the sand gel clogging the syringe. However, while the gel was found to be thick enough to maintain its structure without crosslinking, it ended up collapsing with time. From our findings, we realized that the crosslinking calcium chloride solution would have to be added after the ink has been extruded or layers will not stack on each other to create a 3D structure. On 2025.05.27 Earth Sand Alginate Gel Test (425 and 150 µm), we found that the 50 wt% 425 µm particle size was qualitatively the best for extrusion and layering, though larger structures may need longer cross linking time and/or larger concentration of calcium chloride to reach the same stiffness as smaller structures.
Figure 2. These are the resulting scaffolds after crosslinking in the calcium chloride bath.
Furthermore, though it wasn’t the primary focus, we also found that more than 3 wt% of alginate solution was too insoluble for alginate to dissolve, and instead revised our protocol to set 3 wt% of alginate as our maximum. With the initial trial runs with construction sand from Earth, we finalized a working protocol for making the gel formulation and determined the maximal particle size for extrusion and layering.
Testing with MGS-1
DBTL 1 - MGS-1
We now want to repeat the same experiment using MGS-1 given what was learned using Earth Sand
The same protocol was used, investigating the optimal weight percentage (wt%) of MGS-1 at different wt% and with two different sieve sizes (150 µm, 425 µm).
When first testing the MGS-1 alginate gel, we noted any differences in crosslinking and extrusion of MGS-1 gels compared to Earth Sand gels.
We learned that the MGS-1 alginate gel was not crosslinking readily in the calcium chloride solution as expected.
Figure 2. MGS-1 scaffolds of various particle size submerged in 100 mM calcium chloride solution.
From this DBTL, we observed a lack of noticeable difference between the sieve sizes in terms of resistance and extrusion ease of the MGS-1 alginate gel. Qualitatively, it was observed that the calcium chloride solution alone was not sufficiently diffusing through the regolith to allow crosslinking with the alginate solution and eventual solidification. Thus, the protocol and formulation for MGS-1 alginate gel will need to be troubleshooted and further revised.
DBTL 2 - MGS-1
In order to troubleshoot the crosslinking issues with the MGS-1 alginate gel, we brainstormed a variety of possible issues, such as pH, MGS-1 components, interactions with calcium chloride or CMC, etc. that could be the cause.
For each issue, we devised a protocol to confirm and investigate whether it was interfering with the MGS-1 alginate gel crosslinking.
We carried out a variety of experiments to investigate the issues, mainly comparing the gel scaffolds to the Earth Sand alginate gels, and qualitatively observing whether solidification and crosslinking has improved.
We learned and iterated our protocols following each troubleshooting experiment, which led us to a final range of operable compositions that can form a crosslinked MGS-1 alginate gel
First, we aimed to establish if there is a difference in the pH of MGS-1 bioink in comparison to the Earth sand bioink (2025.07.22 MGS-1 and Earth Sand pH Testing). As it was hypothesized that the perchlorates and salt existing in MGS-1 would cause it to be more alkaline than Earth sand ([9]). However, we found that there was no significant difference in pH of MGS-1 alginate gel in comparison to the Earth sand alginate gel, with both alginate gels having a pH range of 6-7.
Next, we investigated whether calcium diffusion was the issue, where we mixed the calcium chloride solution directly into the alginate gel and compared to a pure alginate control (2025.07.30 Testing MGS-1 Interaction with Calcium Chloride).
Figure 3. Alginate control vs. MGS-1 alginate gel mixed with 100 mM calcium chloride solution. There is noticeable separation of the MGS-1 from the rest of the gel on the right.
Crosslinking was not observed in the 30 wt% MGS-1 and 9 wt% CMC alginate gel despite thoroughly mixing calcium chloride solution in it. On the other hand, the alginate control clumped up indicating sufficient crosslinking.
Thus, the issue was not with calcium diffusion but perhaps other components of the MGS-1 or CMC. Looking at the components in MGS-1 and how they differ in particle size, we wanted to see whether the non-flow through in a sieve size of 150 µm and 425 µm, will affect the crosslinking. This will therefore exclude all components that are finer than the two sieve sizes, as described in Table 2.
Table 2. MGS-1 components by size
Component
% finer than 150µm
% finer than 425µm
Anorthosite
90%
100%
Basalt
40%
70%
Bronzite
20%
65%
Epsomite
NA (water soluble)
NA
Ferrihydrite
100%
100%
Gypsum
50%
95%
Hematite
100%
100%
Hydrated silica
100%
100%
Magnesite
100%
100%
Magnetite
100%
100%
Olivine
>95%
100%
Siderite
100%
100%
Smectite
100%
100%
FIgure 4. Non-flow through (>425µm and >150µm) MGS-1 crosslinking with calcium chloride compared to alginate control.
In 2025.08.01 Isolating MGS-1 Components By Size, MGS-1 was added to alginate and CMC after crosslinking with calcium chloride solution. After addition, the MGS-1 softened the gel. Both the non-flow through conditions, of particle size greater than 150 µm or 425 µm crosslinked better and was stiffer than the flow through seen in 2025.07.15 MGS-1 Alginate Gel Test. However, both conditions were still softer than the alginate control, even though MGS-1 was added after the alginate was already crosslinked.
We then investigated whether MGS-1 is interacting with the other component in the biomaterial, the CMC, since we observed MGS-1 causing the crosslinked alginate and CMC to soften. We wanted to confirm whether MGS-1 itself is disrupting the alginate backbone, resulting in weak crosslinking (2025.08.01 Testing MGS-1 Alginate Gels Without CMC).
Figure 5. Testing whether removing CMC will improve crosslinking.
It was observed that the scaffolds without CMC were stiffer and clumpier than scaffolds with CMC in 2025.07.15 MGS-1 Alginate Gel Test. Scaffolds were able to be picked up and left out to air dry, which could not be achieved before. Thus, we hypothesize that CMC could be interacting with the MGS-1 components, and that the calcium oxide contained within the MGS-1 may be already sufficient to crosslink with the alginate.
We then proceeded with determining the maximum amount of CMC % we can use to still see comparable crosslinking, where the scaffolds were not dissolving and could be picked up. The experiments for this can be found in 2025.08.04 Testing MGS-1 With Varying CMC, 2025.08.06 Recalibrating CMC Composition for MGS-1 Alginate Gel, and 2025.08.08 Finalizing CMC Composition for MGS-1 Alginate Gel. CMC still served as an important component of the bioink, reinforcing the thickness and structure of the alginate gel whilst printing and before adding any calcium chloride to crosslink. However, the current CMC composition at 9wt% was perhaps too high and hindered the ability of MGS-1 to crosslink.
Qualitatively, the operable range of CMC was from 0-4wt% based on observing the scaffolds being able to hold together and be picked up. Anything more than this range would result in a bioink that was more liquid-like, and would not be able to hold up its shape. The operable range of MGS-1 was found to be from 20-40wt%: anything greater than 40wt% was likely too thick for extrusion, and lower than 20wt% lacked structural stability. From follow-up experiments, it was found that the conditions below were able to remain structurally intact after 10 minutes of submerging in 100 mM calcium chloride solution, in order of greater stiffness.
We expected CMC to be able to assist with the extrusion capability of the MGS-1 bio-ink by acting as a thickening agent, but also predicted that a high % of CMC will hinder the cross-linking capabilities of the bio-ink. Thus, we repeated the steps of extruding the compositions shown above in 3 layers to determine which CMC-Alginate composition is best for bioprinting application (2025.08.08 Extrusion and Stacking Test of MGS-1 Alginate Gel). We found that the 3.5% CMC + 20% MGS-1 , 2.5% CMC + 20% MGS-1 , 2% CMC + 30% MGS-1 conditions were able to stack vertically at 3 layers, with the 2.5% CMC + 20% MGS-1 and 2% CMC + 30% MGS-1 conditions being able to maintain a hardened structure upon dehydration.
From this comprehensive DBTL, we troubleshooted all possible issues and found that the operable range of our MGS-1 bioink components in 3wt% alginate that can be cross-linked with 100mM calcium chloride solution was
2-2.5 wt% of CMC
20-30 wt% of MGS-1
DBTL 3 - MGS-1
Now that we have a range of operable bioink components using MGS-1, we wanted to determine which crosslinking method was best for the bioink in order to incorporate into the bioprinter.
We implemented a protocol for introducing calcium chloride through spraying or submerging, and also not submerging the solution at all to see whether the calcium oxide in MGS-1 is sufficient for crosslinking.
We tested the various crosslinking methods in hollow, solid, and grid shapes, noting how many layers can be stacked and whether the scaffolds can be picked up using a spatula.
We learned that not submerging the MGS-1 alginate gels in calcium chloride at all was qualitatively sufficient for crosslinking and allowed more stacking compared to the other methods
Figure 6. Side to side comparison of submerged vs. sprayed vs. unsubmerged method of calcium chloride addition in hollow and solid shapes.
It was qualitatively determined that spraying calcium chloride led to scaffold layers collapsing compared to the other methods. Overall, it was concluded that; (1) Spraying the samples prevented the layers from stacking, (2) Submerging softens the gel but it can still maintain its structure and be picked up and (2) The unsubmerged 3.5% CMC + 20% MGS-1 held up well and did not collapse over 4 hours, with each layer remaining well defined. With these conclusions, we decided to remove the spraying method from consideration and consider not submerging the MGS-1 bioink in calcium chloride at all for sufficient crosslinking.
DBTL 4 - MGS-1
Since EDTA is commonly used as a chelating agent to dissolve cross-linked alginate gels, we hypothesized it potentially having an effect on our bioink structures since BG-11 growth media for UTEX 2973 contains EDTA.
To address this concern, we devised protocols to submerge Earth Sand, MGS-1, and pure alginate gels in various growth mediums, as well as submerge the gels in BG-11 media containing varying EDTA concentration.
We carried out the experiments, noting whether the gels became dissolved or collapsed in varying EDTA concentrations and growth mediums.
We learned that EDTA indeed had an effect on all of the alginate gels, as completing removing this component from BG-11 was the only solution to the MGS-1 alginate gels dissolving.
The growth mediums we tested were PYE media (for CB2A) and BG-11 (for UTEX 2973), mainly concerned with the effects of BG-11, which will be used (2025.08.16 Testing Bioink in BG-11 Media).
Figure 7. Hollow scaffolds of earth sand, MGS-1, and pure alginate gel submerged in PYE or BG-11 media.
It was found that the MGS-1 alginate gel completely degraded in the BG-11 media, while all other inks held their shape in PYE media.
We then proceeded to determine the operable range of EDTA that can be still used in the BG-11 media as it was hypothesized that lower concentrations of EDTA will result in less gel degradation as outlined in (2025.08.19 Testing MGS-1 Alginate Gel in Varying [EDTA] in BG-11 Media).
Figure 8. 3 layered hollow square of 3.5% CMC + 20% MGS-1 bio-ink submerged in 60-100% of the original EDTA concentration in BG-11 media after 1 hour.
Indeed, a high concentration EDTA dissolved alginate as expected due to EDTA disrupting the metal ions in MGS-1 and alginate. Removing EDTA completely allowed the MGS-1 bioink to still maintain its structure. Thus, we proceeded with using BG-11 growth medium without EDTA in order to allow our bioinks to maintain its structure, but the effects of removing EDTA on the growth of UTEX 2973 will need to be further investigated.
It is important to note that the results of these experiments in our DBTLs are purely qualitative and do not contain methodology for quantitative assessment. Hence, future experiments and validations with the incorporation of UTEX 2973 can supplement quantitative analysis to support conclusive and holistic results.
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