Purpose

Repeating 2025.05.26 Earth Sand Alginate Gel Test (850 µm) using sand of 425 and 150 µm particle size.

Materials & Methods

Materials

• 100mM calcium chloride

• 3wt% alginate solution

• Plastic containers for

  • sieved sand
  • mixing the gel

• Magnetic stirrer

• Spatula

• distilled water

• 5mL syringe

• Petri dish Methods

• Used protocol in the methodology section of Bioink Composition Testing

• Calcium chloride solution was added after the scaffolds were extruded

• 5mL gels were made for each condition

• Made 3wt% alginate solution in 55mL DI water

  • added alginate powder into water while mixing on heat

Reagent	Amount Used
CMC	0.45 g
Sand	1.5g, 2g, 2.5g, 3g, 3.5g
Alginate	1.65 g

Results

Figure 1. Particle size of 425 µm (left) and 150 µm (right). Extruded using a straight line then a box with three layers stacked using a 5 mL syringe.

Observations

• Alginate is still very insoluble, needs heat and long time of mixing
• Thickness of the extruded gel seems to affect stiffness
  • the thinner singular string of gel is stiffer (qualitatively) than the 3 layered rectangle
• 150 µm sand (smaller particles) made the gel more soft
• 425 µm sand maintained its shape the best
• Rectangle + line was drawn and traced for each condition to maintain consistent shapes
  • Required only ~2mL of gel

Summary

• More than 3wt% may be too insoluble for alginate to dissolve, set this as max condition and try lower wt%
• 425 µm particle size is qualitatively the best for extrusion + layering
• Sand wt% is inconclusive ---> should play a bigger role in viability and compressibility of bricks
• larger structures may need longer cross linking time / larger concentration of calcium chloride to reach the same stiffness as smaller structures

Hand off

Materials and reagents kept:

• plastics cups
• Sodium alginate (in a resealable bag)
• Figure 1. gel samples
• 1 empty small glass bottle for making alginate solution + 5mL syringes
• 1 empty 1L glass bottle for making CaCl2

Purpose

For diffusion modelling of calcium in the sand alginate gel, we want to obtain a video of calcium chloride solution diffusing as a front to crosslink the alginate on a microscope slide.

Materials & Methods

Materials

• 100 mM calcium chloride
• 3wt% alginate solution
• Earth sand alginate gel (30% 150µm earth sand, 9% CMC, 3% alginate solution, made on 250610)
• Spatula
• 1 mL syringe
• microscope slide + cover slip
• light microscope

Methods

1. To prepare the microscope slide, place 200 μl of the bio-ink (using a 1mL syringe) onto a glass slide and press it with a coverslip. Make sure there are no air bubbles.
2. Mount the slide onto a black surface and below the microscope
3. Start the video recording on your camera.
4. Pipette 200 μl of an aqueous solution of 100 mM calcium chloride. Make sure to do this on ONE side of the coverslip border. Don’t cover the entire sample. The Ca2+ ions will diffuse as a flat front along the hydrogel
5. Let the slide sit and record for 30 minutes.
6. Save the video. It will be used to measure the diffusion distance pixel by pixel.

Results

Figure 1. Microscope slide setup with 200 μl of gel and 200 μl of calcium chloride solution.

Figure 2. No diffusion across was captured. Gel is on the right and the calcium chloride solution was added on the left.

Observations

• Could not see calcium chloride diffusion under microscope (10x objective)

  • Waited ~10 min before attempting to visualize diffusion

• 200 μl of gel seemed like too much or too thick

  • Try 100 μl next to confirm optimal volume
  • Try plating the gel on half of the slide to leave room for an equal volume of calcium chloride on the other half other slide (amount of calcium chloride to add should be slightly larger than the volume of gel added)

• Microscope images taken using iPhone by hand and so it is likely for actual imaging that a better quality microscope with a computer + red laser will be needed

Summary

• Need to optimize the amount of gel on slide vs the volume of calcium chloride on slide to improve quality of measuring diffusion

• Need a higher quality microscope to successfully record calcium chloride diffusion into bioink

• Need to improve earth sand gel formulation to decrease viscosity as thickness seems to be limiting the calcium chloride diffusion process

Hand off

Materials and reagents kept:

• 3wt% alginate solution in the cold room
• 2 microscope slides + 2 cover slips
• 150 μm sieved Earth Sand
• 8mL of 30wt% Earth Sand alginate gel

Purpose

Repeating 2025.06.11 Calcium Diffusion Under Microscope (1) by adding an alginate control to compare against under the microscope.

Materials & Methods

Materials

• 100 mM calcium chloride
• 3wt% alginate solution
• Earth sand alginate gel (30% 150µm earth sand, 9% CMC, 3% alginate solution, made on 250610)
• Spatula
• 1 mL syringe
• microscope slide + cover slip
• light microscope

Methods

1. To prepare the microscope slide, place 200 μl of the bio-ink (using a 1mL syringe) onto a glass slide and press it with a coverslip. Make sure there are no air bubbles.
2. Repeat step 1 but use pure alginate with no CMC or earth sand
3. Mount the slide onto a black surface and below the microscope
4. Start the video recording on your camera.
5. Pipette 200 μl of an aqueous solution of 100 mM calcium chloride. Make sure to do this on ONE side of the coverslip border. Don’t cover the entire sample. The Ca2+ ions will diffuse as a flat front along the hydrogel
6. Let the slide sit and record for 30 minutes.
7. Save the video. It will be used to measure the diffusion distance pixel by pixel.

Results

Figure 3. ~200 μl alginate solution with ~100 μl of calcium chloride added on slide with cover slip (prominent line down the middle). Green arrow indicates the diffusion direction.

Observations

• 100 μl calcium chloride solution was used for 200 μl alginate solution on the coverslip. It seems the calcium chloride solution started to diffuse through the alginate solution under microscope, but we need to wait a longer time to verify

• 40 μl calcium chloride solution was used for 200 μl earth sand alginate gel (30wt% 150 μm sieved sand, 9%CMC, 3% alginate solution) on the coverslip. The earth sand alginate gel did not show any calcium chloride diffusion under the microscope.

Summary

• Calcium chloride diffusion was not observed with 30wt% 150 μm sieved sand, 9%CMC, 3% alginate solution on coverslip

• calcium chloride diffusion observed in alginate control sample, indicating possible issue where earth sand composition is interfering with calcium diffusion ability

Hand off

Materials and reagents kept:

• 3wt% alginate solution in the cold room
• 2 microscope slides + 2 cover slips
• 150 μm sieved Earth Sand

Purpose

For diffusion modelling of calcium crosslinking in the sand alginate gel, we will measure the difference in weight and calculate absorbed calcium chloride volume over different crosslinking times.

Materials & Methods

Materials

• 100 mM calcium chloride
• 3wt% alginate solution
• 30wt% 425 µm earth sand
• 9wt% CMC
• Spatula
• 2 x 1 mL syringe
• 12 x 5 mL glass vials
• red food colouring
• disposable transfer pipette Methods

1. Make 10 mL of 30wt% earth sand, 9wt% CMC and 3wt% alginate gel by following procedure listed in Bioink Composition Testing
2. Load 1 mL of the earth sand gel into a 1 mL syringe and extrude into 6 vials
   1. ensure there are no bubbles, tap the vial down after extruding to ensure the top surface is flat
3. Label each vial with 2, 5, 10, 15, 20, and 60 minutes respectively
4. Repeat steps 1-3 but load pure 3wt% alginate
5. In 15 mL of 100 mM calcium chloride solution, add ~2 drops of red food colouring and mix
6. Weigh each vial and record the initial mass
7. Pipette 1 mL of the red 100 mM calcium chloride solution into each vial
8. Start a timer for crosslinking based on the labelled time
9. Remove the calcium chloride solution using a disposable transfer pipette after 2, 5, 10, 15, 20, and 60 minutes of crosslinking (based on the vial)
   1. ensure not to disturb the gel
10. Weigh the vial again and record the final mass
11. Calculate the volume of calcium chloride absorbed for each time point by using the below formula and a calcium chloride density of 1.007 g/mL
12. Graph crosslinking time vs. volume of calcium chloride absorbed $$\text{Volume}=\frac{\text{Final weight - Initial weight}}{p_{CaCl_2}}$$

Results

Figure 1. 60mins, 20mins, 10mins, 5mins, 2mins (left to right) crosslinking with 30wt% gel. Can see slight pink at the surface going from right to left (with increasing crosslinking time). 15mins is missing since it was taken out.

Figure 2. 60mins, 20mins, 10mins, 5mins, 2mins (left to right) crosslinking with alginate gel. Can see increasing pink / solid gels going from right to left (with increasing crosslinking time). 15mins is missing since I took it out already.

Table 1. Weight measured in grams.

Time (minutes)	Alginate Gel (0%) - initial mass	Alginate Gel (0%) - final mass	Sand + alginate (30%) - initial mass	Sand + alginate (30%) - final mass
2	7.2263	7.2771	7.1635	7.1985
5	7.1541	7.2629	7.2451	7.2798
10	7.1272	7.1893	7.2635	7.3122
15	7.2149	7.2689	7.2608	7.3152
20	7.2552	7.3022	7.0739	7.1381
60	7.1463	7.2284	7.1481	7.2616

Figure 3. Resulting graph for 30wt% earth sand gel

Figure 4. Resulting graph for 0wt% sand, pure 3wt% alginate

Observations

• the alginate gel did not stick to the sides of the vials so calcium chloride solution was diffusing on all sides (not vertical diffusion)
• gels are expanding with time and can see slight pink on the surfaces on the gels with sand
• can see a pink gel after 5 minutes of crosslinking (none for just 2 minutes)

Summary

Seeing a trend of increased volume absorbed (and colour) with increasing crosslinking time

• 5mins may be an outlier for alginate (0% sand), will repeat

• can also calculate mass of CaCl2 absorbed

Hand off

• 4mL left of 30wt% 425 µm earth sand, 9wt% CMC and 3wt% alginate gel in the cold room in the plastic cups

• The obtained data will be fitted against our theoretical diffusion model

Purpose

Testing 30-70 wt% MGS-1 conditions with 425 and 150 µm particle size using methodology from Bioink Composition Testing. Observing whether the gel can be easily extruded from a syringe and maintain its layers after crosslinking.

Materials & Methods

Materials

• 100 mM calcium chloride

• Plastic containers for

  • sieved sand
  • mixing the gel

• Spatula

• 10 mL syringe

• Petri dish

• Deionized water

• 3wt% alginate solution (use prepared one from 2025.06.17)

• Martian regolith (150 µm, 425 µm size)

• Carboxymethyl cellulose (CMC) Methodology

• 5 mL gels were made following methodology from Bioprinter Overview

• 2 sieve sizes * 5 wt% (30, 40, 50, 60, 70) conditions = 10 total tests (no replicates)

• Shapes extruded are shown below

• Calcium chloride solution was added after shapes were extruded

Figure 1. Solid shape (top) and hollow shape (bottom) extruded

Results

Images

150 µm particle size MGS-1

425 µm particle size MGS-1

Image 12. 30-70wt%, 425 µm MGS-1 taken 14-15 hours after calcium chloride submersion.

Observations

• Both sand sizes were easy to extrude through a 5 mL or 10 mL syringe
• The sand solution itself was thick enough for both sieve sizes to allow stacking of layers (alginate solution adds “sticky” texture to bio ink)
• The bioink seem to be disintegrating over time at 150 µm sieve size
  • This is also visible with increasing MGS-1 wt %
• With the 425 µm MGS-1 sieve size, higher wt % seem to hold the layers better
  • This could be due to: 1. larger sand size, 2. change to fresh calcium chloride solution
• Gels were still soft after 14-15 hours of crosslinking for both 150 µm and 425 µm MGS-1 sieve size
  • Attempts to move the bio ink with a spatula resulted in the structure falling a part
• Bio ink lightened in colour gradually after calcium chloride submersion
• The 40 wt% 425 µm regolith appeared to be slightly more solidified than the 70 wt% regolith
  • Both structures were still very soft and unable to be lifted from the plate
  • suggest lower wt % which can provide better calcium chloride diffusion and ultimately better diffusion

Summary

• There was no noticeable difference between the sieve sizes in terms of resistance, extrusion ease, or printability of bio ink
• Any “potential solidification” observed likely linked to the sand “stickiness” rather than calcium chloride crosslinking with alginate solution
• The calcium chloride solution alone is not sufficiently diffusing through the regolith to allow crosslinking with the alginate solution and eventual solidification

1. Literature will need to be investigated to determine current strategies for MGS-1 used in building materials
2. Current bio ink production protocol will need to be optimized for MGS-1 conditions
3. A repeat of this experiment must be conducted to ensure proper protocol design

• The layers of bioink held together much better for the 150 µm compared to the 425 µm regolith
  • 3 layers are more distinguishable for 150 µm
  • The layers collapsed on each other for 425 µm

Hand off

• New calcium chloride stock prepared
• Need to make new batch of alginate solution
• Restocked CMC
• Some 450 µm sieved MGS-1 leftover

Purpose

Determining if there is a difference in the pH of MGS-1 or Earth sand, which could interfere with crosslinking as seen in 2025.07.15 MGS-1 Alginate Gel Test.

Materials & Methods

Materials

• 2g of Earth sand

• 2g of MGS-1

• DI water

• pH litmus paper Methods

• In 2 beakers, pipette 100 mL of DI water into each

• Add in 2g of Earth sand in one beaker and 2g of MGS-1 in the other

• Mix for 2 minutes and let sit for 10 minutes

• Use the litmus paper to determine the pH

Results

Observations

• no observable difference between earth sand and MGS-1

• both sands settled at the bottom after 10 minutes

• no visible change in colour of the litmus paper in both conditions

Summary

• pH is neutral for both MGS-1 and earth sand (no difference) → not the issue for crosslinking

Hand off

• no gels were made / used

Purpose

Autoclaving 50 wt% earth sand alginate gel and mineralization medium to prepare for UTEX 2973 incorporation and mineralization testing.

Materials & Methods

Materials

• 2x 100 mL bottles

• 10 mL syringes

• Plastic containers for

  • 425 µm sieved earth sand
  • 9 wt% CMC

• 3 wt% alginate solution

• stock BG-11 media components

• MilliQ water

• sodium bicarbonate

• calcium chloride Methods

• Made 50 mL of 50wt%, 425 µm earth sand alginate gel in an 100 mL bottle

  • autoclave using LIQUID20 setting

• Made 100 mL mineralization BG-11 medium

  • Add 50 mL MilliQ water to a clean 100 mL bottle
  • Add the BG-11 media components below, except calcium chloride

  Component	Volume added (uL)
  1000x MgSO4	100
  1000x K2HPO4	100
  1000x CaCl2	100
  1000x Na2CO3	100
  1000x ferric ammonium citrate + citric acid	100
  1000x Na2EDTA	100
  1000x BG-11 trace metals	100
  100x NaNO3	1000

  • top up with MilliQ water to 100 mL
  • slowly add 1.1 g of calcium chloride (100 mM) and 0.84 sodium bicarbonate
  • autoclave using LIQUID20 setting

Results

• Mineralization medium formed precipitate immediately

• Earth sand alginate gel overflowed after autoclaving

Summary

• 50 mL of gel in a 100 mL bottle was too much volume

  • Try a smaller volume, 10 mL in a 100 mL bottle
  • Also try separately autoclaving each bioink component to allow more flexibility in making different wt% gels (alginate, CMC, and sand separately)

• concentrations of sodium bicarbonate and calcium chloride are too high since carbonate crystals are already forming # Hand off

• Autoclaved mineralization medium

• 20 mL of 3wt% alginate in 4C room

Purpose

Autoclaving 50 wt% earth sand alginate gel (in a smaller amount) and variations of mineralization medium to prepare for UTEX 2973 incorporation and mineralization testing.

Materials & Methods

Materials

• 3x 100 mL bottles

• 10 mL syringes

• Plastic containers for

  • 425 µm sieved earth sand
  • 9 wt% CMC

• 3 wt% alginate solution

• stock BG-11 media components

• MilliQ water

• sodium bicarbonate

• calcium chloride Methodology

• Made 10 mL of 50wt% 425 µm earth sand alginate gel in a 100 mL bottle

  • autoclaved on LIQUID 20 setting

• Made 2 variations of mineralization media

  • BG-11 media + 100 mM sodium bicarbonate + 100 mM calcium chloride
    • add 100 mL MilliQ water to 100 mL bottle
    • add 0.84g of sodium bicarbonate
    • add 1.1g of calcium chloride
    • add rest of BG-11 components from Table 1 except calcium chloride
  • BGS-11 media + 100 mM sodium carbonate + 100 mM calcium chloride
    • add 100 mL MilliQ water to 100 mL bottle
    • add 1.06g of sodium carbonate
    • add 1.1g of calcium chloride
    • add rest of BG-11 components from Table 1 except calcium chloride and sodium carbonate

Table 1. BG-11 Components for 100 mL

Component	Volume added (uL)
1000x MgSO4	100
1000x K2HPO4	100
1000x CaCl2	100
1000x Na2CO3	100
1000x ferric ammonium citrate + citric acid	100
1000x Na2EDTA	100
1000x BG-11 trace metals	100
100x NaNO3	1000

• Prepare 30 mL of 3wt% alginate solution to autoclave on LIQUID20 setting
  • add calcium chloride to a sample of autoclaved pure alginate to see whether it can still crosslink
• Prepare CMC and 425 µm sieved sand in separate beakers to autoclave on dry cycle

Results

Observations

• precipitates formed when calcium chloride and sodium bicarbonate/carbonate are added together

  • dissolves completely when components are added alone

• Earth sand gel successfully autoclaved and still crosslinks with calcium chloride addition

• Pure alginate still crosslinks with calcium chloride addition after autoclaving (did not dissolve or disintegrate)

• CMC and sieved earth sand also autoclaved on dry cycle

Summary

• autoclaving is a suitable sterilization method for the bioink components (does not interfere with crosslinking)

• precipitates still form with variations of mineralization medium, will proceed with curing the gels in the prepared mineralization medium and just BG-11 growth medium (without addition of calcium chloride and carbonate)

Hand off

• all reagents prepared in this experiment can be used to incorporate live UTEX 2973

• when opening these reagents, use sterile technique (open under a flame)

Purpose

Testing the protocol for incorporating wild type UTEX 2973 into 50 wt% 425 µm earth sand, 9 wt% CMC, and 3 wt% alginate gel, as well as curing it in mineralization medium to test for calcium carbonate formation.

Materials & Methods

Materials

• UTEX 2973 liquid culture (‘Passage 5’)
• Cuvettes for OD check
• 1 mL syringes
• 50 wt%, 425 µm earth sand alginate gel
• 35 mm petri dishes
• BG-11 + 100 mM calcium chloride + 100 mM sodium bicarbonate mineralization medium
• BG-11 growth medium
• BG-11 + 100 mM calcium chloride + 100 mM sodium carbonate mineralization medium
• Falcon/microcentrifuge tubes
• Centrifuge Methods

1. Take OD750 reading of ‘Passage 5’ Culture using the spectrophotometer

2. Determine cell concentration (cells/mL) using the calibration equation (x = OD)

   $$y=\left(10^{6.34}\right)\left(x\right)^{\left(0.9\right)}$$

3. Determine how many mL (y) of culture needed for 1g of bioink ([1])

   $$\frac{7\cdot10^{7}}{y}$$

4. Transfer the amount of culture needed for 1g of bioink into an appropriately sized falcon tube

5. Centrifuge at 3700g for 10 minutes (until bacterial pellet is prominent)

6. Discard supernatant and resuspend pellet in 100 µL BG-11 growth medium

7. Load 1 mL of gel into a sterile syringe

8. Transfer bacteria resuspension into a sterile microcentrifuge tube and add the 1 mL of gel

9. Mix well by aspirating up and down with a syringe

10. Load the bioink into the syringe and extrude into a petri dish:

    1. hollow square shape
    2. 3x3 grid shape

11. Submerge scaffolds in sterile 100 mM calcium chloride solution and allow it to crosslink for 10 minutes

12. Remove calcium chloride solution with a pipette

13. Submerge scaffolds in 2 mL mineralization medium

14. Parafilm the opening of the petri dish and place into the light incubator (maintained between 30-38ºC)

Results

Observations

• 4 samples

  • Grid shape: BG11 only and BG11 + 100mM CaCl2 + 100mM Na2CO3
  • Rectangle shape: BG11 only and BG11 + 100mM CaCl2 + 100mM NaHCO3

• 1 mL of bioink weight was approximately 1.31 g

• 2 x 40 mL Passage 5 culture was loaded in a 50 mL falcon tube (OD750 was 0.80, ~38 mL needed for 70 million cells)

• Bacterial pellet contained a top green layer and white/yellow bottom layer

  • bottom layer may be contamination and/or dead cells

• Final resuspension was ~ 300 µL

• 1 mL of bioink made 2 shapes

• Tried to use an 18G needle to extrude the shape, but was clogged by the sand

• Precipitate began accumulating around the scaffold when submerged in the two mineralization mediums

• After 1 day, the grid scaffold in BG-11 + calcium chloride + sodium carbonate dissolved/disintegrated

• On day 4, grids began to bleach and lose its green colour

Summary

• a lot of bacteria / suspension volume is required for 1 mL of bioink → consider lowering seeding density

• mixing bacteria suspension with the bioink was quite inefficient, consider using syringe adaptors to mix better

• need to determine viability of cells prior to incorporation and after certain incubation times in the media

  • consider doing a live/dead stain

Hand off

• 4 small petri dishes of 3 different medium conditions stored in the light incubator

Purpose

Troubleshooting why the MGS-1 alginate gel is not crosslinking (observable solidification) after submerging in 100 mM calcium chloride. This experiment involves mixing calcium chloride directly into the MGS-1 alginate gel as that would inform whether the calcium is diffusing and reacting with the alginate at all.

Materials & Methods

Materials

• Plastic container for mixing the MGS-1 alginate gel
• DI water
• 10 mL syringes
• 100 mM calcium chloride solution
• 3 wt% alginate solution
• 425 µm sieved MGS-1
• CMC
• spatula Methods

1. Make the control alginate sample
   1. Add 10 mL of 3 wt% alginate solution to a plastic container
   2. Add 9 wt% (0.9g) of CMC to the solution and mix
   3. Add 10 mL of calcium chloride to the alginate gel mixture
   4. Let sit for 10 minutes to observe clumping / solidification
2. Make the MGS-1 alginate gel sample
   1. Add 10 mL of 3 wt% alginate solution to a plastic container
   2. Add 9 wt% (0.9g) of CMC to the solution and mix
   3. Add 30 wt% (3 g) of 425 µm sieved MGS-1 to the solution and mix
   4. Add 10 mL of calcium chloride solution to the MGS-1 gel mixture
   5. Let sit for 10 minutes to observe clumping / solidification, using the control sample as a reference

Results

Observations

• 9 wt% of CMC formed clumps when mixing into the alginate gel
• alginate control solidified after adding 1 mL of calcium chloride (gels clumped and pulled apart in pieces)
  • increasingly solidified with increasing calcium chloride solution
• crosslinking was not observed in MGS-1 alginate gel when calcium chloride was mixed in
  • gel became more dilute and liquid-like with increasing amounts of calcium chloride
  • there was noticeable separation of MGS-1 from the rest of the gel (settling at the bottom)

Summary

• crosslinking was not observed in alginate gel containing MGS-1 despite thoroughly mixing calcium chloride in it

• pure alginate crosslinked without issue, perhaps indicating other components, such as the MGS-1 itself or CMC is interfering with the crosslinking

Hand off

• new alginate solution and calcium chloride were made

Purpose

Troubleshooting why the MGS-1 alginate gel is not crosslinking (observable solidification) after submerging in 100 mM calcium chloride. This experiment involves isolating certain components of martian regolith by size to determine whether certain components are disrupting the crosslinking by also testing 150 and 425 µm non-flow through MGS-1. The components of the non-flow through are listed in Bioink Composition Testing.

Materials & Methods

Materials

• plastic containers

• 150 µm sieved MGS-1 + non-flow through (>150µm) MGS-1

• 425 µm sieved MGS-1 + non-flow through (>425µm) MGS-1

• 3wt% alginate solution

• 100 mM calcium chloride

• 10 mL syringe

• petri dish

• 9 wt% CMC Methods

• Sieve the MGS-1 in 150 and 425 µm sieve sizes, saving the non-flow through

• Make 5 mL of 30 wt% MGS-1 and 9wt% CMC alginate gels for each condition (2)

  • < 150 µm and > 425µm

• Extrude 1 mL of gel and pure alginate control into a petri dish and submerge in 100 mM calcium chloride solution

• Let crosslink for 10 minutes

• Mix non-flow through with pre-crosslinked alginate to observe whether the crosslink disintegrates

Results

Observations

• Both sieve sizes (>150µm and >425µm) crosslinks but not as well as the alginate control
• when MGS-1 is added to the pre-crosslinked alginate, both > 150µm and > 425µm softened the gel

  • 150 µm was stiffer than the >425 µm

Summary

• Using the non-flow through improved crosslinking compared to 2025.07.15 MGS-1 Alginate Gel Test but not to the same extent as alginate gel control

• Discarding flow-through may be non-representative of MGS-1 as a simulant of martian regolith

Hand off

• 150 µm and 425 µm sieved regolith is kept

Purpose

Troubleshooting why the MGS-1 alginate gel is not crosslinking (observable solidification) after submerging in 100 mM calcium chloride. This experiment involves removing CMC from the gel composition and studying the effects of MGS-1 on alginate only.

Materials & Methods

Materials

• 425 µm sieved MGS-1

• 3 wt% alginate solution

• petri dishes

• plastic container

• 100 mM calcium chloride solution

• spatula Methods

• Set up 8 petri dishes contained 1 mL of 3 wt% alginate gel solution

• Add 0g, 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g (0-70wt% respectively) of 425 µm sieved MGS-1 to the alginate

• Mix homogeneously using a spatula

• submerge in 3 mL of 100 mM calcium chloride solution for up to 30 minutes

• Qualitatively measure the interaction using the spatula by probing and moving the scaffold in the bath

• Remove the samples from calcium chloride and let dry

Results

Figure 1. 3 wt % alginate + 0-70 wt% MGS-1 submerged in 100 mM calcium chloride.

Figure 2. 3 wt % alginate + 0-70 wt% MGS-1 dried for 8 hours.

Observations

• no observations of alginate degradation in the presence of MGS-1

• All crosslinked better than with CMC in 2025.07.15 MGS-1 Alginate Gel Test

• Wt% of 10-40% MGS-1 showed similar level of crosslinking and resistance to 0% MGS-1 (pure alginate) control

• Above 50 wt%, scaffolds broke into individual clumps

• small clumps form prior to crosslinking for all conditions that includes MGS-1

Summary

• Proceed with 10-40wt% of MGS-1

• alginate and MGS-1 interacting alone is similar to just alginate and earth sand

• formation of clumps may indicate that calcium existing in the MGS-1 is crosslinking with the alginate

  • CMC may be interfering with this interaction

Hand off

• Next will be determining the operable range of CMC% that will still provide desirable crosslinking

Purpose

Repeating the alginate control from 2025.06.21 Calcium Diffusion in Vials (Earth Sand) to obtain better experimental data with less outliers.

Materials & Methods

Refer to 2025.06.21 Calcium Diffusion in Vials (Earth Sand)

• only alginate (with no earth sand) will be tested

Results

Table 1. Weight measured in grams.

Time (minutes)	Alginate Gel (0%) - initial mass	Alginate Gel (0%) - final mass
2	7.1788	7.1978
5	7.15	7.1729
10	7.1496	7.1763
15	7.1651	7.2207
20	7.1644	7.259
60	6.9964	7.1313

Figure 1. Resulting graph of calcium chloride volume absorbed vs. crosslinking time.

Observations

• the alginate gel did not stick to the sides of the vials so calcium chloride solution was diffusing on all sides (not vertical diffusion)

• can see a pink gel after 5 minutes of crosslinking (none for just 2 minutes)

Summary

• trend of increased volume absorbed + colour with increasing crosslinking time

Hand off

• Fit this data to theoretical diffusion model of calcium and compare against earth sand alginate gel

Purpose

Troubleshooting why the MGS-1 alginate gel is not crosslinking (observable solidification) after submerging in 100 mM calcium chloride. This experiment involves testing varying wt % of CMC with different wt % of MGS-1 to determine what range of CMC allows for comparable crosslinking.

Materials & Methods

Materials

• 425 µm sieved MGS-1
• 3 wt% alginate solution
• CMC
• 2 x 12-well plates
• 100 mM calcium chloride solution
• spatula Methods

1. Measure weight needed for 1 mL 10-60wt% 425 µm MGS-1, at 2-9wt% CMC alginate gels in 12-well plates and add to each well
2. Extrude 1 mL of 3wt% alginate solution into each well
3. mix homogeneously with a spatula
4. Gather the bioink into a spherical shape and submerge in 100 mM calcium chloride for 10 minutes

Results

Figure 1. All conditions after mixing, before submerging in 100 mM calcium chloride.

Summary

• Operable range for CMC is 0-4wt%. All conditions more than this resulted in a more liquid-like scaffold

• Weight % greater than 40% for MGS-1 resulted in clumps that were hard to mix and extrude. This is likely due to calcium in the MGS-1 crosslinking with the alginate.

Hand off

• All samples are left to air dry

Purpose

After finding the operable range of CMC, we aim to find a suitable concentration of CMC and MGS-1 that creates a gel that is liquid enough to extrude smoothly, but at the same time be viscous enough to hold layers on itself.

Materials & Methods

Materials

• 425 µm sieved MGS-1
• 3 wt% alginate solution
• CMC
• 2 x 12-well plates
• 100 mM calcium chloride solution
• spatula
• 10 mL syringe Table 1. Amount of CMC/MGS-1 weighed per wt%

CMC/MGS-1 wt%	20	30	40
0	0g/0.4g	0g/0.6g	0g/0.8g
0.5	0.01g/0.4g	0.01g/0.6g	0.01g/0.8g
1	0.02g/0.4g	0.02g/0.6g	0.02g/0.8g
1.5	0.03g/0.4g	0.03g/0.6g	0.03g/0.8g
2	0.04g/0.4g	0.04g/0.6g	0.04g/0.8g
2.5	0.05g/0.4g	0.05g/0.6g	0.05g/0.8g
3	0.06g/0.4g	0.06g/0.6g	0.06g/0.8g
3.5	0.07g/0.4g	0.07g/0.6g	0.07g/0.8g
4	0.08g/0.4g	0.08g/0.6g	0.08g/0.8g

Methods

• Add 2 mL 3wt% alginate to a plastic container
• Add CMC and MGS-1, mix wel
• Transfer gel to a 12-well plate
• submerge in 100 mM calcium chloride for 10 minutes
• Leave overnight

Results

Figure 1. Gel consistencies before crosslinking.

Observations

• with increasing time of mixing (without crosslinking), the gel became thicker

• samples with 0wt% CMC solidified before submerging in calcium chloride

• 0.5wt% CMC + 30wt% MGS-1 has similar pre-crosslinking consistencies to 4wt% CMC + 30wt% MGS-1. Both are very soft.

• No pattern seen with same CMC percentages across different regolith concentrations, vice versa

Summary

• consistently seeing that the gel is solidifying before submerging in calcium chloride - MGS-1 and alginate is crosslinking itself

• CMC reduces this crosslinking between MGS-1 and alginate

• The below conditions was qualitatively the best in terms of thickness and be able to hold structure after crosslinking with calcium chloride

  • 2.5 wt% CMC + 20wt% MGS-1
  • 3.5 wt% CMC + 20 wt% MGS-1
  • 2wt% CMC + 30wt% MGS-1
  • 0.5wt% CMC + 40wt% MGS-1
  • 2wt% CMC + 40wt% MGS-1
  • 3.5wt% CMC + 40wt% MGS-1

Hand off

• the six conditions above will be tested once again to finalize and rank the MGS-1 alginate gel composition

Purpose

With the finalized CMC composition in the MGS-1 alginate gel, we wanted to compare and determine if they can be extruded and stacked onto 3 layers.

Materials & Methods

Materials

• 425 µm sieved MGS-1

• 3 wt% alginate solution

• CMC

• 2 x 12-well plates

• 100 mM calcium chloride solution

• spatula

• 10 mL syringe Methods

• Made 5 mL MGS-1 3 wt% alginate gels of conditions below

2.5% CMC + 20% MGS-1
3.5% CMC + 20% MGS-1
2% CMC + 30% MGS-1
0.5% CMC + 40% MGS-1
2% CMC + 40% MGS-1
3.5% CMC + 40% MGS-1

• follow the methodology listed in Bioink Composition Testing to extrude a hollow rectangle of three layers and three lines

Results

Observations

• all conditions were able to stack up to 6 layers

  • up to 6 layers, structure began to be unstable

• all conditions held structure after overnight drying

• there was lots of air in the syringe that can cause inconsistent extrusion

Summary

• try spraying calcium chloride between layers to aid in cross-linking

  • may help with structures collapsing by having surface of layers crosslinked, but spraying may add excess liquid that can cause the structure to fall apart

• results can be carried over to response surface modelling for bioink optimization

• CMC should be in the range of approximately 1-4 wt%

Hand off

• results can be carried over to response surface modelling for bioink optimization

Purpose

From 2025.08.06 Recalibrating CMC Composition for MGS-1 Alginate Gel, recreate the six best conditions with replicates to determine the most suitable MGS-1 alginate gel. This will be based on qualitatively observing the crosslinking and stiffness after submerging in calcium chloride, as well as compare to an earth sand alginate gel control

Materials & Methods

Materials

• 425 µm sieved MGS-1
• 3 wt% alginate solution
• CMC
• 2 x 12-well plates
• 100 mM calcium chloride solution
• spatula
• 10 mL syringe Methods

1. Add 2 mL alginate to a well in the 12-well plate
2. Add CMC and MGS-1, mix well
3. Add CMC and earth sand (for the same conditions as MGS-1)
4. Submerge in 100mM CaCl2 (~1 mL)
5. Wait 10min to see if crosslinking occurs
6. Create 4 replicates of each condition. Repeat for all other conditions
7. Leave overnight Table 1. Amount to weigh for CMC and MGS-1.

Condition	CMC (g)	MGS-1 (g)
2.5% CMC + 20% MGS-1	0.05	0.4
3.5% CMC + 20% MGS-1	0.07	0.4
2% CMC + 30% MGS-1	0.04	0.6
0.5% CMC + 40% MGS-1	0.01	0.8
2% CMC + 40% MGS-1	0.04	0.8
3.5% CMC + 40% MGS-1	0.07	0.8

Results

Figure 1. MGS-1 alginate gels before submerging in calcium chloride (left). Earth Sand alginate gel controls in calcium chloride (middle). MGS-1 alginate gles in calcium chloride (right).

Observations

• all conditions are soft before crosslinking

  • increasing wt% in CMC led to softer gels
  • able to extrude through a syringe

• control samples crosslinked quickly (surface was stiff after 10 minutes in calcium chloride)

• all samples held better after 10 minutes in calcium chloride

Summary

• At 10min of calcium chloride crosslinking, the order of greater stiffness/crosslinking is:

1. 3.5% CMC + 20% MGS-1
2. 2.5% CMC + 20% MGS-1
3. 2% CMC + 30% MGS-1
4. 0.5% CMC + 40% MGS-1 = 2% CMC + 40% MGS-1 = 3.5% CMC + 40% MGS-1

Hand off

• test extrusion + layering with the above conditions

Purpose

To test whether spraying calcium chloride solution works for crosslinking on pure alginate, earth sand, and MGS-1 alginate gels. Scaffolds were compared to submerging in calcium chloride for 10 minutes.

Materials & Methods

Materials

• square petri dish (for sprayed samples)

• 5 mL syringe

• 100 mM calcium chloride

• 3 wt% alginate

• spatula

• 35mm petri dish (for submerged samples) Methods

• Make 5 mL alginate gels of the following:

  • 3 wt% alginate
  • 3 wt% alginate + 3.5 wt% CMC + 20 wt% MGS-1 (425um)
  • 3 wt% alginate + 9 wt% CMC + 30 wt% earth sand (425um)

• Spraying method

  • Extrude a single layer (square)
  • spray 3x with 100mM calcium chloride solution
  • immediately extrude the next layer
  • repeat 3 times
  • let sit for 10 mins then try picking up the scaffold to see if it had solidified

• Submerge method

  • Extrude 3-5 layers (square)
  • Submerge with 100 mM calcium chloride solution
  • let sit for 10 mins then try picking up the scaffold to see if it had solidified

Results

Observations

• For Earth sand alginate gel, layers could not stick even if extruding the next layer right after the spray

  • layers are visible in submerged bath but still collapsing

• For submerged MGS-1 alginate gel, it could be picked up with spatula after 10 minutes of crosslinking

• the MGS-1 alginate gel was clumpy before extrusion

• The sprayed MGS-1 alginate gel could not be picked up and was easily broken apart

Summary

• Spraying calcium chloride does not work for alginate / earth sand alginate gel → the layers do not stick or stack

• MGS-1 alginate gels seem to hold layers well without calcium chloride (but not solidified), adding calcium chloride makes its softer

  • unsubmerging may be considered sufficient crosslinking

Hand off

• for each finalized CMC composition with MGS-1, make 5 mL alginate gels for each condition and try not submerging, submerging, and spraying with calcium chloride to compare and determine the best method of crosslinking

Purpose

To test the top three MGS-1 alginate gel compositions in hollow and filled rectangular shapes to see whether the spraying or submerging method of calcium chloride crosslinking is best.

Materials & Methods

Materials

• 3wt% alginate

• square petri dish (for spray method)

• 35mm petri dish (for submerged method)

• 5mL syringes

• 100mM calcium chloride

• 425 um MGS-1

• CMC Methods

• Make 5 mL of MGS-1 alginate gels for each formulation

	3wt% alginate + 3.5% CMC + 20% MGS-1	3wt% alginate + 2.5% CMC + 20% MGS-1	3wt% alginate + 2% CMC + 30% MGS-1
Alginate (mL)	5	5	5
CMC (g)	0.175	0.125	0.1
MGS-1 (g)	1	1	1.5

• Extrude the MGS-1 alginate gels and then do the following:
  • three layered hollow square

1. no addition of calcium chloride bath
2. spray 100 mM calcium chloride three times between each layer (extrude the next layer immediately) → try to remove scaffold and leave on paper towel to dry
3. submerge scaffold in 100 mM calcium chloride after extruding for 10 minutes → remove scaffold from bath and leave on paper towel to dry
   • three layered rectangular brick
4. no addition of calcium chloride bath
5. spray 100 mM calcium chloride three times between each layer (extrude the next layer immediately) → try to remove scaffold and leave on paper towel to dry
6. submerge scaffold in 100 mM calcium chloride after extruding for 10 minutes → remove scaffold from bath and leave on paper towel to dry

Results

Figure 3. All samples removed from 100 mM calcium chloride and left out on the lab bench to dry.

Observation

• Submerged samples

  • hollow square layers were most visible and shape was retained
  • solid shape held its shape and height, but inside was not crosslinked

• Sprayed samples

  • hollow square held shape but was difficult to stack
  • layers became separated
  • much softer compared to the submerged samples

• more CMC led to more layers being able to hold up

• extrusions were smooth

• samples began to crosslink, so needed to be filled into syringe quickly (add MGS-1 last)

Summary

• 3.5wt% CMC + 20wt% MGS-1 layers help up vertically and crosslinked better than the others

• Mix MGS-1 with alginate thoroughly before putting it in the syringe, quality of extrusion goes down otherwise

• Spraying the samples prevents layers from stacking. Sprayed structures behave more like a wound string than a brick

• Unsubmerged 3.5% CMC + 20% MGS-1 held up well and did not collapse over 4 hours. Each layer remained well defined

Hand off

• samples are left on the lab bench to dry

Purpose

To test whether different formulations of alginate gels (earth sand and MGS-1) degrade in PYE media (CB2A) and BG-11 media (UTEX 2973). This will inform whether the growth medium recipes need to be adjusted for curing the alginate gels.

Materials & Methods

Materials

• PYE media (per 2025.05.21 Preparation of PYE Broth)

• BG-11 media (per BG-11 Medium Recipe)

• Petri dishes

• 425 um MGS-1 and Earth Sand

• CMC

• 3wt% alginate

• 100 mM calcium chloride Methods

• Make 5 mL alginate gels (2 of each) of the following:

	3wt% alginate	3wt% alginate + 3.5% CMC + 20% MGS-1	3wt% alginate + 9% CMC + 30% Earth Sand
Alginate (mL)	5	5	5
CMC (g)	0	0.175	0.45
MGS-1/ Sand (g)	0	1	1.5

• Extrude four layer structures into petri dish using a 5 mL syringe
• Submerge alginate control and Earth sand alginate gel in 100 mM calcium chloride for ten minutes
  • leave MGS-1 alginate gel unsubmerged
• Remove calcium chloride from the dish
• Submerge each gel formulation in:
  • PYE media
  • BG-11 media

Results

Figure 1. Scaffolds submerged in respective growth medium.

Observations

• 10 minutes after submerging, MGS-1 alginate gel was degrading in the BG-11 media
  • all other alginate gels held their shape
• after 24 hrs, MGS-1 alginate gel completely degraded
  • all other alginate gels held their shape

Summary

• current MGS-1 alginate gel composition completely dissolves in BG-11 media → need to research components and determine what may be the cause

• need to come up with a working BG-11 media composition to allow UTEX 2973 to grow and be incorporated in MGS-1 alginate gel

Hand off

• samples are parafilmed and left to incubate in room temperature

Purpose

To determine what working concentration of EDTA, up to 1000x the concentration of EDTA in current BG-11 media recipe, would allow the MGS-1 alginate gel to not dissolve. We hypothesize that lower concentrations of EDTA will result in less bioink degradation.

Materials & Methods

Materials

• small petri dishes

• 3 wt% alginate

• 425 um sieved MGS-1

• 5 mL syringes

• metal spatula

• BG-11 media components (per BG-11 Medium Recipe) Methods (adapted from BG-11 Medium Recipe)

• In a 100 mL bottle, add 100 mL MilliQ water, then ad the BG-11 components except EDTA

• Take out 10 mL of the above media and transfer to a falcon tube (repeat six times)

• Each 10 mL in a falcon tube represents a % of sodium EDTA in the original BG-11, add the following amounts according to the %

% of Na2EDTA in original BG-11	Volume added (uL)
0%	0
20%	2
40%	4
60%	6
80%	8
100%	10

• make 30 mL of 3.5 wt% CMC + 20 wt% MGS-1 3wt% alginate gel
• Fill a 5 mL syringe with the alginate gel and extrude into petri dish of a hollow square with three layers
• submerge in BG-11 medium of varying EDTA concentrations
• Prepare another sample where the MGS-1 alginate gel is submerged in stock [EDTA] (1000x the concentration in BG-11)
• extrude 1 mL of 3 wt% alginate onto a petri dish
  • submerge in 100 mM calcium chloride for 10 minutes to crosslink
  • remove calcium chloride and submerge in 1 mL of 1000x EDTA

Results

Figure 1. MGS-1 alginate gel submerged in 1 mL BG-11 media of varying [EDTA] after 1 hour.

Observations

• 40% and higher EDTA concentrations would disintegrate the MGS-1 alginate gel

• EDTA also dissolved pure alginate, but at a much slower rate than MGS-1 alginate gel

• only 0% EDTA showed a structure that still held up after one day

• when BG-11 media removed from 40% petri dish and replaced with 100 mM calcium chloride, the MGS-1 alginate gel crosslinked and became stiff

Summary

• when curing the gels with UTEX 2973, the BG-11 media will not include sodium EDTA to prevent the gel from collapsing and disintegrating

• 1000x [EDTA] also dissolve alginate - as expected since EDTA is meant to dissolve alginate gels

• EDTA is perhaps disrupting metal ions in MGS-1 alginate gels

Hand off

• BG-11 media without sodium EDTA made

Purpose

To validate whether UTEX can grow without the presence of environmental oxygen by keeping the bacterial culture in an anaerobic chamber (consisting of only carbon dioxide, hydrogen, and nitrogen gas). Oxygen is predicted to be used in metabolic reactions of UTEX 2973, but as an autotrophic organism, it is still able to sustain biomass formation without environmental oxygen.

Materials & Methods

Materials

• Anaerobic Chamber

• PBS

• MilliQ water

• Disposable cuvettes

• parafilm

• kimwipes

• spectrophotometer

• liquid culture of UTEX

• 500 mL culture flask

• sterile glass culture tubes

• BG-11 media Methods

• Day 0

  • Take the culture flask out of the incubator and aseptically take 1 mL of culture into a clean cuvette under the flame
  • Set spectrophotometer to OD750 and blank with BG-11 media
  • Wipe cuvette body with kimwipe
  • place the cuvette into the spectrophotometer and measure initial OD
  • aliquot 5 mL of culture into a sterile glass culture tube
  • place the tube into the anaerobic chamber
  • move culture tubes onto shaker at a speed of 200 rpm and cover with light box

• Day 2

  • remove culture tubes from the anaerobic chamber and take an OD measurement following the same protocol
    • if OD is not within an appropriate range of 0.1 and 1, dilute the sample with PBS

• Day 4

  • remove culture tubes from the anaerobic chamber and take an OD measurement following the same protocol
    • if OD is not within an appropriate range of 0.1 and 1, dilute the sample with PBS

• Day 7

  • remove culture tubes from the anaerobic chamber and take an OD measurement following the same protocol
    • if OD is not within an appropriate range of 0.1 and 1, dilute the sample with PBS

Results

Table 1. OD750 measurements

Date	Time Checked (h)	Culture Time (h)	Dilution Factor	Measured OD	True OD
18-Sep-25	9:35 PM	PBS (blank)	1	-0.001	0
18-Sep-25	9:35 PM	0	1	0.089	0.088
20-Sep-25	7:55 AM	34	1	0.117	0.116
22-Sep-25	7:15 AM	70	1	0.454	0.453
25-Sep-25	9:20 PM	168	5	0.272	1.355

Figure 1. OD750 of UTEX 2973 over 7 days.

Summary

• UTEX 2973 is able to sustain growth (linear trend) in anaerobic conditions

Hand off

• culture flask is removed from chamber and bleached

Purpose

For diffusion modelling of calcium crosslinking in the MGS-1 alginate gel, we will measure the difference in weight and calculate absorbed calcium chloride volume over different crosslinking times.

Materials & Methods

Materials

• 100 mM calcium chloride
• 3wt% alginate solution
• 20wt% 425 µm MGS-1
• 3.5wt% CMC
• Spatula
• 2 x 1 mL syringe
• 12 x 5 mL glass vials
• red food colouring
• disposable transfer pipette Methods

1. Make 10 mL of 20wt% earth sand, 3.5wt% CMC and 3wt% alginate gel by following procedure listed in Bioink Composition Testing
2. Load 1 mL of the MGS-1 gel into a 1 mL syringe and extrude into 6 vials
   1. ensure there are no bubbles, tap the vial down after extruding to ensure the top surface is flat
3. Label each vial with 2, 5, 10, 15, 20, and 60 minutes respectively
4. Repeat steps 1-3 but load pure 3wt% alginate
5. In 15 mL of 100 mM calcium chloride solution, add ~2 drops of red food colouring and mix
6. Weigh each vial and record the initial mass
7. Pipette 1 mL of the red 100 mM calcium chloride solution into each vial
8. Start a timer for crosslinking based on the labelled time
9. Remove the calcium chloride solution using a disposable transfer pipette after 2, 5, 10, 15, 20, and 60 minutes of crosslinking (based on the vial)
   1. ensure not to disturb the gel
10. Weigh the vial again and record the final mass
11. Calculate the volume of calcium chloride absorbed for each time point by using the below formula and a calcium chloride density of 1.007 g/mL
12. Graph crosslinking time vs. volume of calcium chloride absorbed $$\text{Volume}=\frac{\text{Final weight - Initial weight}}{p_{CaCl_2}}$$

Results

Figure 1. Vials containing 1 mL MGS-1 gel and 1 mL red calcium chloride solution

Table 1. Weight measured in grams.

Time (minutes)	MGS-1 gel initial mass	MGS-1 gel final mass
2	7.1689	7.1956
5	7.1273	7.1598
10	7.2991	7.3387
15	7.2609	7.3018
20	7.2654	7.3165
60	7.2181	7.2703

Figure 2. Resulting graph for MGS-1 gel calcium chloride volume absorbed.

Observations

• Volume of calcium chloride absorbed is significantly less (20-60 µL) compared to up to 100 µL in earth sand and alginate gels from 2025.06.21 Calcium Diffusion in Vials (Earth Sand)

• 20 mins vs 60 mins crosslinking have very minimal volume difference

• Red solution observed on the surface of the gel, but no visible diffusion

Summary

• Seeing a trend of increased volume absorbed with increasing crosslinking time

• No significant difference in volume absorbed in 20 mins vs 60 mins crosslinking, suggesting that longer than 20 minutes of submerging in calcium chloride may not make a difference in crosslinking

• MGS-1 composition may be hindering calcium diffusion as volume absorbed is a lot less than earth sand and pure alginate gels

Hand off

• The obtained data will be fitted against our theoretical diffusion model

Purpose

To test whether adding commercial carbonic anhydrase (CA) to a 3 wt% alginate / 3.5% CMC bioink containing 20 wt% MGS-1 (Martian regolith simulant) accelerates carbonate mineralization (CaCO₃) and increases gel cross-linking/mechanical strength when exposed to Ca²⁺ and CO₂. This will serve as a positive control and demonstrate that we can produce bricks using this bioink.

Materials & Methods

Materials

• bioink (3% alginate, 3.5%CMC, 30wt% MGS-1(425um))

• 3 wt% alginate solution

• CMC

• 425 µm sieved MGS-1

• Sigma Aldrich Carbonic Anhydrase from bovine erythrocytes

• Calcium Chloride solution

• 6-well plates

• erlenmeyer flasks

• carbon dioxide pump / chamber (uses carbon dioxide from dry ice) Methods

• Make 10 mL each of:

  • 3 wt% alginate + 3.5 wt% CMC + 5 mg CA
  • 3 wt% alginate + 3.5 wt% CMC + 20 wt% MGS-1 + 5 mg CA

• Add CA to the dry mix and add to the alginate solution

• Extrude three shapes of each condition in a 6-well plate

  • straight lines
  • grid shape
  • hollow rectangle

• crosslink with 100 mM calcium chloride solution for 10 minutes

• connect the experimental setup to the carbon dioxide pump

  • switch to more concentrated calcium chloride solution if needed
  • place in a ziploc bag to ensure minimal carbon dioxide escapes
  • ensure the tubes are submerged in the solution and that carbon dioxide is directly bubbled in

• let sit for up to 24 hours or when crystallization is observed

Results

Figure 1. Scaffolds submerged in 100 mM calcium chloride in 6-well plate (left). Plate connected to the carbon dioxide pump, was difficult to get individual tubes bubbling carbon dioxide separately into the wells (middle). Final experimental setup connected to the carbon dioxide pump, with all scaffolds submerged in 1M calcium chloride solution in a flask (right).

Figure 2. Scaffolds removed from carbon dioxide pump and calcium chloride solution after incubating for 24 hours (left). Scaffolds air dried for 24 hours in 37C (right). The middle row consists of MGS-1 gels containing carbonic anhydrase, while the bottom row does not contain any carbonic anhydrase.

Figure 3. Light microscope image of the alginate gel containing carbonic anhydrase after 24 hours incubation in carbon dioxide pump and calcium chloride solution. Freckles and granular view on the bottom half may be calcium carbonate precipitate.

Observations

• only a singular tube was on the carbon dioxide pump → put all scaffolds into a singular flask to better ensure sufficient carbon dioxide delivery

• the 3 wt% alginate and CMC control could not hold its shape and could not form the hollow or grid structure (collapsed and filled the entire well)

• MGS-1 alginate gel could be picked up by a tweezer and not fall apart after 10 minutes crosslinking in 100 mM calcium chloride

• Used 1M calcium chloride solution in the flask

• the 3 wt% alginate and CMC control became cloudy and could see microbubbles after 30 minutes in the bubbler and 1M calcium chloride solution

• scaffolds were removed from the solution after ~24 hrs and left out to air dry for another 24 hrs

  • scaffolds were like a stiff gel

• Dried MGS-1 alginate samples were qualitatively comparable in strength to non-CA samples from previous experiments

  • though this is not conclusive as the non-CA samples were left out for very long

• The alginate control scaffolds were opaque and rough to touch

Summary

• qualitatively, there is a difference in touch and transparency in the alginate gels when incorporating carbonic anhydrase

• opaque and granular view on the microscope may be indicating the precipitation of calcium carbonate

• need a more quantifiable method in comparing the compressibility between gels containing CA, and those than do not