R.K.B. acknowledges support from the Presidential Postdoctoral Research Fellows Program. This material is also based upon work supported by NSF Graduate Research Fellowship Program Grant DGE-2039656 (to A.M.H.). A.S.D.-M. and H.N.L. acknowledge support from the Lidow Independent Work/Senior Thesis Fund at Princeton University. We also thank the laboratory of Bonnie Bassler for providing strains of V. cholerae. S.S.D. acknowledges support from NSF Grants CBET-1941716, DMR-2011750, and EF-2124863, as well as the Eric and Wendy Schmidt Transformative Technology Fund, the New Jersey Health Foundation, the Pew Biomedical Scholars Program, and the Camille Dreyfus Teacher-Scholar Program.

This approach is to convert a commercial 3D fused deposition modeling printer into a 3D bioprinter using a previously established protocol by Tashman et al.34. In brief, Tashman et al. replaced a commercial extruder head with a custom-made syringe pump extruder. This extruder enables the printing of highly concentrated liquid suspensions of bacterial cells in 3D, with its extruded volume and 3D position controlled by the G-code programming language. The extruded volume is specified in the software by the extruder step (E-step) and is additionally calibrated as described further below. These bacterial suspensions are thereby printed directly into a granular hydrogel matrix, which acts as a 3D support for the cells. Below, the protocol also describes how to prepare matrices with different polymer concentrations, characterize the resulting changes in pore size and rheological properties, and characterize subsequent bacterial motility and growth using direct imaging.
1. Conversion of a commercial 3D printer into a 3D bioprinter
<ol>
	<li>Remove the extruder and the heater from a commercial 3D printer (see Table of Materials).</li>
	<li>Follow previous protocols to fabricate the syringe pump extruder34, with an additional modification to accommodate a disposable Luer lock syringe. Mount the syringe pump extruder onto the printer. 
	NOTE: The CAD files required for modifying the syringe pump extruder for plastic syringes are provided in&#160;Supplementary Files 1-3.</li>
	<li>Install and open the 3D printer software (see Table of Materials) on a computer. Connect the 3D printer to the computer.</li>
	<li>Load a 1 mL disposable syringe with an appropriately sized needle into the 3D-printed clamps by aligning the top and bottom halves of the mechanism (Figure 1). Secure the clamps around the syringe using three M8 socket bolts and thin steel hex nuts (see Table of Materials). The syringe plunger connects with the lead screw in the syringe pump extruder. Manually raise the plunger by rotating the lead screw to create an 0.5 mL of air gap in the syringe.</li>
	<li>If contamination is a concern for the experiment, transport the syringe-clamp complex to a biohood and sterilize with 70% ethanol spray upon entry before completing the following steps.</li>
</ol>
2. Preparation of the bacterial suspension
<ol>
	<li>For V. cholerae and E. coli, grow&#160;overnight on a 2% Lennox LB (Luria Broth, see Table of Materials) agar plate at 37 &#176;C.
	<ol>
		<li>For V. cholerae, inoculate the cells into 3 mL of liquid LB with ten sterile glass beads. Grow the cells in a shaking incubator at 37 &#176;C for 5-6 h until the mid-exponential phase to an optical density (OD) 600 of ~0.9.</li>
		<li>For E. coli, inoculate the cells into 3 mL of liquid LB. Grow the cells in a shaking incubator at 37 &#176;C overnight. Innoculate 200 &#181;L of the overnight culture in fresh LB for 3 h until the OD reaches 0.6.</li>
	</ol>
	</li>
	<li>Transfer the culture into a 10 mL centrifuge tube. Centrifuge the culture for 5 min at 5,000 x g at room temperature to form a pellet. Remove the supernatant. Resuspend with ~10 &#181;L of liquid LB to achieve a cell density of ~9 x 1010 cells per mL.</li>
</ol>
3. Loading the bacterial suspension into the syringe
NOTE: Two methods are provided for loading the bacteria into the syringe (step 3.1 and step 3.2). Step 3.1 works for loading small volumes of bacterial suspensions, &#60;200 &#181;L, and step 3.2 works for loading larger volumes of bacterial suspensions, &#62;200 &#181;L. Step 3.1 was utilized for the representative results shown here.
<ol>
	<li>Load an empty 1 mL plastic Luer lock syringe into the 3D bioprinter. Connect the syringe plunger with the lead screw. Manually retract the syringe to add 0.2 mL of the air gap to provide space for the plunger to move in the syringe as a small volume of cells ~20-50 &#181;L are used for each batch of experiments.
	<ol>
		<li>Attach a blunt needle to the syringe tip with the needle size necessary for the size of print features required. Here, a 2-inch 20 G needle is used.</li>
		<li>Load the bacterial suspension into the syringe by placing a 10 mL centrifuge tube with the bacterial inoculum below the needle. Manually rotate the screw to retract the syringe plunger and load the cells into the syringe. The bacterial cell volumes are so small that often, the cells are only loaded into the needle.</li>
	</ol>
	</li>
	<li>Remove the plunger from the syringe-clamp complex and use another syringe and needle to carefully load the syringe-clamp complex with the desired bacterial suspensions, being careful to avoid trapping air bubbles. The syringe-clamp complex should be filled slightly over the brim with the desired bacterial suspension and then transferred to the bioprinter.
	<ol>
		<li>Carefully insert the syringe-clamp complex without the plunger into the corresponding socket on the main core of the bio-printer extruder.</li>
		<li>Ensure that the printer carriage is approximately halfway up the lead screw, and a collection dish is present under the loaded syringe. Then, carefully insert the plunger through both the carriage and the syringe-clamp complex until it catches on the carriage. Depress the plunger slowly into the bacterial suspension to avoid trapping air bubbles in the syringe.</li>
		<li>Slide the adaptor clamp onto the carriage over the back of the plunger to secure it in place for both extrusion and retraction maneuvers.</li>
	</ol>
	</li>
</ol>
4. Calibration of the extruder step to deposited volume
<ol>
	<li>To calibrate the extruder step (E-step) to the deposited volume, first set up the bio-printer with the exact syringe, syringe needle, and depositing bacterial suspension that will be used in the experiment. Here, a 1 mL Luer lock syringe is used.</li>
	<li>Determine an estimated E-step range to calibrate over by extruding an arbitrary E-step number (~200) and note the plunger volume change with syringe volume markers.</li>
	<li>Use this coarse E-step to volume ratio to determine the E-step settings to perform the calibration sweep over. For example, if an E-step of 200 extrudes approximately 20 &#181;L by visual inspection and one wants to deposit 10-200 &#181;L, test E-steps between 100-2000.</li>
	<li>To perform the linear calibration sweep, first label and measure the dry mass of twenty 1.5 mL sampling tubes on an analytical balance with 0.1 mg sensitivity.</li>
	<li>Extrude bacterial suspension into the pre-measured 1.5 mL tubes. For each E-step, perform at least 2 replicates. Repeat for all E-steps over the linear range, replacing bacterial suspension as necessary. If contamination is a concern for the experiment, wipe the exterior of the syringe needle with a lint-free wipe saturated with 70% ethanol after each sample.</li>
	<li>Measure the mass of all 1.5 mL tubes with the same analytical balance. Subtract the first mass value from the second to obtain a net mass of bacterial suspension deposited.</li>
	<li>Convert the mass of bacterial suspension into a volume with the material density. For many bacterial suspensions composed primarily of water, 1 g/mL is an appropriate density approximation.</li>
	<li>Perform a linear fit between the E-step and the extruded volume to finish the calibration process.</li>
</ol>
5. Preparation of the granular hydrogel matrix
<ol>
	<li>In a biosafety cabinet, add dry granules of cross-linked acrylic acid/alkyl acrylate copolymers (see Table of Materials) to 400 mL of 2% Lennox Luria-Bertani (LB) to keep the matrix sterile; however, other liquid cell culture media can also be used to swell the hydrogel matrix. 
	NOTE: The weight percent of granules added to the LB depends on the pore size that one is aiming for. In the present study, for a 0.9% granular hydrogel matrix, 3.6 g of dry granules are added to the LB and for a 1.2% granular hydrogel matrix, 4.8 g of dry granules are added to the LB. The hydrogel granules are homogenously dispersed by mixing them in a stand mixer for 2 min.</li>
	<li>Once mixed, adjust the pH to 7.4 by adding 20 to 500 &#181;L increments of 10 M sodium hydroxide (NaOH) to ensure cell viability. After each addition of NaOH, measure the pH by dipping a pipette tip into the mixture and then wiping the hydrogel matrix onto a pH test paper. 
	NOTE: As the NaOH is added, the viscosity of the mixture will increase as the hydrogel granules start to swell. The swollen hydrogel granules are ~5 &#181;m to 10 &#181;m in diameter and jammed together in a hydrogel matrix. The internal mesh size of the granules is ~40 nm to 100 nm, as previously established32. The mesh size is large enough for small molecules (e.g., oxygen and nutrients) to freely diffuse but small enough for bacteria to be confined between the interstitial pores.</li>
	<li>Next, transfer the granular hydrogel matrix to a 50 mL centrifuge tube using a 50 mL sterile plastic syringe. Centrifuge the hydrogel matrix at 161 x g for 1 min at room temperature to remove the bubbles formed during the mixing process.</li>
	<li>Allow the hydrogel matrix to sit for at least 2 days at room temperature to ensure that no contamination has occurred. The contamination appears as microcolonies suspended in the hydrogel matrix. After two days, centrifuge the hydrogel matrix at 161 x g for 1 min to remove any additional bubbles that formed. 
	NOTE: The protocol can be paused here by storing the hydrogel matrix at room temperature for up to a week.</li>
	<li>In the biosafety cabinet, using a 30 mL sterile plastic syringe, transfer the desired amount of hydrogel matrix to the container where printing will occur (here ~20 mL for a 20 mL tissue culture flask or 1 mL for 1 mL plastic micro cuvettes were used).</li>
</ol>
6. Characterization of the granular hydrogel matrix rheological properties
<ol>
	<li>Load ~3 mL of the hydrogel matrix into a shear rheometer (see Table of Materials) with a 1 mm gap between roughened 50 mm diameter parallel plates to measure the rheological properties.</li>
	<li>Quantify the yield behavior using unidirectional shear measurements on the shear rheometer by measuring the shear stress as a function of a logarithmic sweep of shear rate from 10-4 s-1 to 102 s-1 (e.g., Figure 2A). 
	NOTE: At low shear rates, the hydrogel matrix will have a constant shear stress (the yield stress) that is independent of the shear rate. At high shear rates, the shear stress will increase with a power-law dependence on the shear rate, indicating the fluidizing of the hydrogel matrix. This yield-stress behavior allows bacteria to be 3D printed within the granular hydrogel matrix29.</li>
	<li>Measure the storage and loss moduli, G&#39; and G&#39;&#39; respectively, as a function of frequency using small amplitude oscillatory rheology with a strain amplitude of 1% and frequencies between 0.1 to 1 Hz (e.g., Figure 2B). 
	NOTE: The ideal granular hydrogel matrix for 3D printing should have a storage modulus larger than the loss modulus, which indicates the medium acts as a jammed elastic solid29.</li>
</ol>
7. Characterization of the granular hydrogel matrix interstitial pore size
<ol>
	<li>Sonicate 100 nm carboxylated fluorescent polystyrene nanoparticles (~3.6 x 1013 particles/mL, see Table of Materials) in their packaging for 15 min to resuspend to break up any aggregations/clusters of particles. Transfer 50 &#181;L of nanoparticles to a 1.5 mL microcentrifuge tube.
	<ol>
		<li>Centrifuge for 10 min at 9,500 x g at room temperature until the pellet forms and the supernatant is clear. Remove the supernatant and resuspend the pellet in 1 mL of the liquid growth media (here LB) used to prepare the granular hydrogel matrix. 
		NOTE: The protocol can be paused here by storing the resuspended nanoparticles at 4 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>Sonicate the resuspended nanoparticles for 30 min. Transfer 1 mL of granular hydrogel matrix to a 1.5 mL microcentrifuge tube. Add 1 &#181;L of the resuspended nanoparticles to the granular hydrogel matrix and mix them with a pipette tip. After mixing, centrifuge for 30 s at 161 x g at room temperature.</li>
	<li>Transfer the hydrogel matrix and the nanoparticle mixture to a 35 mm diameter Petri dish with a 0.1 mm thick glass bottom well. The well is 20 mm in diameter and 1 mm in depth. Place a glass coverslip on top and press down to disable flow and evaporation during imaging. An alternative to the glass coverslip is to add 1 mL of paraffin oil on the top.</li>
	<li>Image the nanoparticles using a confocal microscope with a 40x oil objective with 8x additional zoom in the imaging software (see Table of Materials). 
	NOTE: A higher magnification objective could be used instead of using additional digital zoom in the software.
	<ol>
		<li>Image a time loop with no delay (ideally ~19 frames/s) in a single z-plane for 2 min with at least four nanoparticles within the field of view. Repeat 15 to 20 times in different locations to collect enough data for statistics (100 to 200 nanoparticles).</li>
	</ol>
	</li>
	<li>Use a particle tracking software to analyze the displacement of the particles. Here, a custom-written script is used based on the classic Crocker-Grier algorithm to track the center of mass of the nanoparticle35 (see Supplementary File 7).
	<ol>
		<li>From the particle tracking, calculate the mean square displacement (MSD). The MSD will exhibit free diffusion in the pore space at short lengths and time scales and transition to sub-diffusive scaling at large lengths and time scales due to confinement35.</li>
	</ol>
	</li>
	<li>Identify the length scale where the transition to sub-diffusive scaling occurs to estimate the local pore size. Calculate pore size by adding this length scale to the nanoparticle diameter. Repeat the pore size analysis for every nanoparticle measured. This will yield a pore size distribution from which a mean pore size can be calculated (e.g., Figure 3).</li>
</ol>
8. 3D printing process
<ol>
	<li>3D-print custom-made holders for the sample containers (see Supplementary Files 4-6 for the CAD files). Here, holders for the tissue culture flasks and microcuvettes are used. The holders allow for programming the printer to print multiple samples in a single print session. Place the sample containers with hydrogel media in the holders on the build platform.</li>
	<li>Open the 3D printing software. Load a pre-programmed g-code into the software. For the representative results, step 3.1 is used to load the bacterial suspension into the 3D printer. 
	NOTE: An example of a g-code for printing linear vertical geometries is given in Table 1.</li>
	<li>Through the 3D printing software, move the x-y-z planes to center the print head on the x-y plane to be the first container and then home the z-axis. The homing z-axis will raise the print head. Manually rotate the screw slowly to depress the syringe plunger until a small amount of the bacterial suspension can be seen at the tip of the needle.
	<ol>
		<li>Lightly wipe the excess bacterial suspension off with a sterile disposable wipe. Based on the height of the sample holder, needle, and syringe, using the 3D printing software, lower the print head a fixed distance into the hydrogel media in the sample container of choice. Start the printing process by clicking on Print.</li>
	</ol>
	</li>
	<li>Once the printing is complete, close the sample containers. Wipe down the printer with 70% ethanol. Properly dispose of the syringe and needle.</li>
</ol>
9. Growing and imaging the V. cholerae
<ol>
	<li>For large field-of-view imaging, use a camera with a zoom lens attachment to image cell growth with a lightbox. Image the samples right after printing at room temperature and then transfer them to an incubator. Maintain samples at 37 &#176;C in a stationary incubator between imaging sessions during the experiment.</li>
	<li>Capture images over a desired amount of time to observe the growth behavior at long periods in the granular hydrogel matrix.</li>
</ol>

Bioprinting Bacteria in Hydrogel Matrices for the Study of Motility and Growth

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+semi-solid+agar+for+the+detection+of+bacterial+motility.">The use of semi-solid agar for the detection of bacterial motility.</a> Journal of Bacteriology. 31 (6), 575-580 (1936).</li><li>Bhattacharjee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+scaling+laws+for+microgel+yielding+near+jamming.">Polyelectrolyte scaling laws for microgel yielding near jamming.</a> Soft Matter. 14 (9), 1559-1570 (2018).</li><li>Bhattacharjee, T., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confinement+and+activity+regulate+bacterial+motion+in+porous+media.">Confinement and activity regulate bacterial motion in porous media.</a> Soft Matter. 15 (48), 9920-9930 (2019).</li><li>Bhattacharjee, T., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+hopping+and+trapping+in+porous+media.">Bacterial hopping and trapping in porous media.</a> Nature Communications. 10 (1), 2075 (2019).</li><li>Bhattacharjee, T., Amchin, D. B., Ott, J. A., Kratz, F., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+migration+of+bacteria+in+porous+media.">Chemotactic migration of bacteria in porous media.</a> Biophysical Journal. 120 (16), 3483-3497 (2021).</li><li>Bhattacharjee, T., Amchin, D. B., Alert, R., Ott, J. A., Datta, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+smoothing+of+collective+migration.">Chemotactic smoothing of collective migration.</a> eLife. 11, e71226 (2022).</li><li>Tashman, J. W., Shiwarski, D. J., Feinberg, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+performance+open-source+syringe+extruder+optimized+for+extrusion+and+retraction+during+FRESH+3D+bioprinting.">A high performance open-source syringe extruder optimized for extrusion and retraction during FRESH 3D bioprinting.</a> HardwareX. 9, e00170 (2021).</li><li>Crocker, J. C., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+digital+video+microscopy+for+colloidal+studies.">Methods of digital video microscopy for colloidal studies.</a> Journal of Colloid and Interface Science. 179 (1), 298-310 (1996).</li><li>Chatterjee, T., Chatterjee, B. K., Chakrabarti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+growth+kinetics+of+Vibrio+cholerae+in+presence+of+gold+nanoparticles:+Effect+of+size+and+morphology.">Modelling of growth kinetics of Vibrio cholerae in presence of gold nanoparticles: Effect of size and morphology.</a> Scientific Reports. 7 (1), 9671 (2017).</li><li>Mart&#237;nez-Calvo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+instability+and+roughening+of+growing+3D+bacterial+colonies.">Morphological instability and roughening of growing 3D bacterial colonies.</a> Proceedings of the National Academy of Sciences. 119 (43), e2208019119 (2022).</li><li>Lehner, B. A. E., Schmieden, D. T., Meyer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Straightforward+approach+for+3D+bacterial+printing.">A Straightforward approach for 3D bacterial printing.</a> ACS Synthetic Biology. 6 (7), 1124-1130 (2017).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+cell+ordering+in+confined+bacterial+biofilms.">Morphogenesis and cell ordering in confined bacterial biofilms.</a> Proceedings of the National Academy of Sciences. 118 (31), e2107107118 (2021).</li></ol>

The experimental platform used to 3D print and image bacterial communities in this publication is the subject of a patent application filed by Princeton University on behalf of Tapomoy Bhattacharjee and S.S.D. (PCT Application number PCT/US/2020/030213).

Critical steps in the protocol 
It is important to ensure that when preparing each hydrogel matrix, the matrix is made in a sterile environment. If not, contamination can occur, which manifests as, e.g., microcolonies (small spheroids) in the matrix after several days. During the mixing process, it is important that all the dry granular hydrogel particles are dissolved. Additionally, when adjusting the pH of each hydrogel matrix with the NaOH, the granules will start to swell, which increases the viscosity of the hydrogel matrix, leading to mixing being more difficult. Using the stand mixer will help ensure that the NaOH is well mixed into the hydrogel matrix. During the loading of each bacterial suspension, air pockets can form in the needle. To avoid this issue, ensure that the needle tip is always sitting in the bacterial suspension in the centrifuge tube and not at the bottom of the tube or near the top surface. Another way to overcome this issue is to grow large volumes of cells and thus have larger volumes of the bacterial suspension for printing.
Limitations 
Currently, during printing, the low viscosity of the bacterial suspension limits the geometries that can be printed and often leads to a biofilm-forming and growing on the top of the hydrogel matrix surface due to trace cells. There are a few potential methods for overcoming this limitation, including increasing the viscosity of the bacterial suspension or further optimizing the 3D printer settings. To increase the viscosity of the bacterial suspension, one could mix the bacterial suspension with another polymer - for example, alginate, which has been used prior for the 3D printing of bacteria onto flat surfaces38. The printer settings can be further optimized to enable retraction of the syringe plunger during the withdrawal of the needle from the granular hydrogel matrix, which would have the potential to stop cells from being deposited during the removal of the needle from the hydrogel matrix.
The significance of the method with respect to existing/alternative methods 
The method described here allows for the printing of bacterial colonies into granular hydrogel matrices. The granular hydrogel matrices allow the study of the impact of external environmental factors (e.g., pore size, matrix deformability) on the motility and growth of bacteria. Additionally, while in this work, LB is used as the liquid growth medium to swell the hydrogel matrix, the hydrogel matrix can be swollen with other liquid growth media, including media with antibiotics. Previous methods for studying bacteria in confined environments were limited by the length of experimental time, the polymer mesh size, and surrounding hydrogel matrix stiffness37,38. Protocols already exist for making granular hydrogel matrices out of different polymers, so the potential for studying the impacts of different environmental conditions on the motility and growth of bacteria is vast. This method allows for the study of bacteria in control environments that more readily recapitulate the environments that bacteria inhabit in the real world, such as host mucus or soil. Another limitation of many other methods is the opacity of the surrounding matrix; however, this approach using optically transparent materials provides the ability to explore, e.g., optogenetic control and patterning of bacteria in 3D.
Beyond studying motility and growth, the 3D printing method described here overcomes the limitation of many other bioprinting methods that require the deposition of a bioink on a substrate and are, therefore, limited in the height of the engineered living material they can produce. In the future, this bioprinting protocol can be further expanded to fabricate biohybrid materials by mixing polymers with biofilm-forming cells. The granular hydrogel matrices provide support for 3D printing thicker, larger-scale engineered living materials and more complex geometries than many other current bacteria bioprinting methods. While this work only used V. cholerae and E. coli, other species, such as Pseudomonas aeruginosa, have also successfully been 3D printed37. Beyond printing, the printer can be adapted to do a controlled sampling of bacteria after growth to see if there have been any genetic changes, for example.

Bacteria often inhabit diverse, complex 3D porous environments ranging from mucosal gels in the gut and lungs to soil in the ground1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21, 
22,23,24,25. In these settings, bacterial movement through motility or growth can be impeded by surrounding obstacles, such as polymer networks or packings of solid mineral grains-influencing the ability of the cells to spread through their environments26, access nutrient sources, colonize new terrain, and form protective biofilm communities27. However, traditional lab studies typically employ highly simplified geometries, focusing on cells in liquid cultures or on flat surfaces. While these approaches yield key insights into microbiology, they do not fully recapitulate the complexity of natural habitats, leading to dramatic differences in growth rates and motility behavior compared to measurements performed in real-world settings. Therefore, a method to define bacterial colonies and study their motility and growth in 3D porous environments more akin to many of their natural habitats is critically needed.
Inoculating cells into an agar gel and then visualizing their macroscopic spreading by eye or using a camera provides one straightforward way to accomplish this, as first proposed by Tittsler and Sandholzer in 193628. However, this approach suffers from a number of key technical challenges: (1) While the pore sizes can, in principle, be varied by varying the agarose concentration, the pore structure of such gels is poorly defined; (2) Light scattering causes these gels to be turbid, making it difficult to visualize cells at the individual scale with high resolution and fidelity, particularly in large samples; (3) When the agar concentration is too large, cell migration is restricted to the top flat surface of the gel; (4) The complex rheology of such gels makes it challenging to introduce inocula with well-defined geometries.
To address these limitations, in previous work, Datta&#39;s lab developed an alternate approach using granular hydrogel matrices - comprised of jammed, biocompatible hydrogel particles swollen in liquid bacterial culture - as &#34;porous Petri dishes&#34; to confine cells in 3D. These matrices are soft, self-healing, yield-stress solids; thus, unlike with cross-linked gels used in other bioprinting processes, an injection micronozzle can move freely inside the matrix along any prescribed 3D path by locally rearranging the hydrogel particles29. These particles then re-densify rapidly and self-heal around injected bacteria, supporting the cells in place without any additional harmful processing. This process is, therefore, a form of 3D printing that enables bacterial cells to be arranged - in a desired 3D structure, with a defined community composition - within a porous matrix having tunable physicochemical properties. Moreover, the hydrogel matrices are completely transparent, enabling the cells to be directly visualized using imaging.
The utility of this approach has been demonstrated previously in two ways. In one set of studies, dilute cells were dispersed throughout the hydrogel matrix, which enabled studies of the motility of individual bacteria30,31. In another set of studies, multicellular communities were 3D-printed in centimeter-scale gels using an injection nozzle mounted on a programmable microscope stage, which enabled studies of the spreading of bacterial collectives through their surroundings32,33. In both cases, these studies revealed previously unknown differences in the spreading characteristics of bacteria inhabiting porous environments compared to those in liquid culture/on flat surfaces. However, given that they were mounted on a microscope stage, these previous studies were limited to small sample volumes (~1 mL) and, therefore, short experimental time scales. They were also limited in their ability to define inocula geometries with high spatial resolution.
Here, the next generation of this experimental platform that addresses both limitations is described. Specifically, protocols are provided by which one can use a modified 3D printer with an attached syringe extruder to 3D print and image bacterial colonies at large scales. Moreover, representative data indicates how this approach can be useful for studying the motility and growth of bacteria, using the biofilm-former Vibrio cholerae and planktonic Escherichia coli as examples. This approach enables bacterial colonies to be sustained over long times and visualized using various imaging techniques. Hence, the ability of this approach to study bacterial communities in 3D porous habitats has tremendous research and applied potential, impacting the treatment and study of microbes in the gut, the skin, the lung, and the soil. Moreover, this approach could be used in the future for 3D printing bacteria-based engineered living materials into more complex freestanding shapes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL cuvettes</td>
			<td>VWR</td>
			<td>97000-586</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Luer lock syringe</td>
			<td>BH Supplies</td>
			<td>BH1LL</td>
			<td></td>
		</tr>
		<tr>
			<td>10 M NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>72068</td>
			<td></td>
		</tr>
		<tr>
			<td>100 nm carboxylated fluorescent polystyrene nanoparticles (FluoSpheres)&#160;</td>
			<td>&#160;Invitrogen, (ThermoFischer Scientific)</td>
			<td> 
			F8803</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>ThermoFischer Scientific</td>
			<td>14-955-237</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G blunt needle</td>
			<td>McMaster Carr</td>
			<td>75165A252</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL tissue culture flasks</td>
			<td>VWR</td>
			<td>10861-566</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Lulzbot</td>
			<td>LulzBot Mini 2</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printing software</td>
			<td>Cura</td>
			<td>Cura-Lulzbot</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>ThermoFischer Scientific</td>
			<td>14-955-239</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbomer Granular Hydrogel Particles</td>
			<td>Lubrizol&#160;</td>
			<td>Carbopol 980NF</td>
			<td>dry granules of crosslinked acrylic acid/alkyl acrylate copolymers</td>
		</tr>
		<tr>
			<td>Centrifuge (2 mL tube capacity)</td>
			<td>VWR</td>
			<td>2405-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (50 mL tube capacity)</td>
			<td>ThermoFischer Scientific</td>
			<td>75007200</td>
			<td>Sorvall (brand) ST 8 (model)</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>A1R+ inverted laserscanning 
			confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petri dish</td>
			<td>Cellvis</td>
			<td>D35-10-1-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Lennox LB (Lubria Broth)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>M8 &#215; 1.25 mm, 150 mm long, Fully Threaded Socket Cap</td>
			<td>McMaster Carr</td>
			<td>91290A478</td>
			<td></td>
		</tr>
		<tr>
			<td>M8 &#215; 1.25 mm, Brass Thin Hex Nut</td>
			<td>McMaster Carr</td>
			<td>93187A300</td>
			<td></td>
		</tr>
		<tr>
			<td>Open-source syringe pump</td>
			<td>Custom-made</td>
			<td>Replistruder 4</td>
			<td>https://www.sciencedirect.com/science/article/pii/S2468067220300791</td>
		</tr>
		<tr>
			<td>Petri dish (60 mm round)</td>
			<td>ThermoFischer Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Shear Rheometer</td>
			<td>Anton Paar</td>
			<td>MCR 501</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>VWR</td>
			<td>97043-992</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d printing bacteria to study motility and growth in complex 3d porous media

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. However, the majority of previous work has focused on studies of cells in bulk liquids or at flat surfaces, which do not fully recapitulate the complexity of many natural bacterial habitats. Here, this gap in knowledge is addressed by describing the development of a method to 3D-print dense colonies of bacteria into jammed granular hydrogel matrices. These matrices have tunable pore sizes and mechanical properties; they physically confine the cells, thus supporting them in 3D. They are optically transparent, allowing for direct visualization of bacterial spreading through their surroundings using imaging. As a proof of this principle, here, the capability of this protocol is demonstrated by 3D printing and imaging non-motile and motile Vibro cholerae, as well as non-motile Escherichia coli, in jammed granular hydrogel matrices with varying interstitial pore sizes.

Watch this Scientific Journal Video about Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices at JoVE.com

Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes.

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. ...

Bacteria are ubiquitous in complex, three-dimensional, porous environments such as biological tissues and gels and subsurface soils and sediments. Here we develop a method to 3D print dense colonies of bacteria into jammed granular hydrogel matrices to study their growth and motility in complex environments. Studies have revealed previously unknown differences in spreading characteristics of bacteria inhabiting porous environments compared to those in liquid cultures on flat surfaces.The development of using granular hydrogel matrices comprised of jammed biocompatible hydrogel particles swollen in liquid bacterial culture as porous Petri dishes to confine cells in 3D. Previous studies were limited to small sample volumes about one ml, and therefore short experimental time scales, and were also limited in their ability to define inocula geometries with high spatial resolution. We have established that the cell-spreading characteristics of the bacteria depend on the pore size and the motility of the cell.

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2.0K Views.  University of Colorado Boulder. Bacteria are ubiquitous in complex, three-dimensional, porous environments such as biological tissues and gels and subsurface soils and sediments. Here we develop a method to 3D print dense colonies of bacteria into jammed granular hydrogel matrices to study their growth and motility in complex environments. Studies have revealed previously unknown differences in spreading characteristics of bacteria inhabiting porous environments compared to those in liquid cultures on flat surfaces.T...

Video: Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...