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.
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.
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's lab developed an alternate approach using granular hydrogel matrices – comprised of jammed, biocompatible hydrogel particles swollen in liquid bacterial culture – as "porous Petri dishes" 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.
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.
The authors have nothing to disclose.
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.
1 mL cuvettes | VWR | 97000-586 | |
1 mL Luer lock syringe | BH Supplies | BH1LL | |
10 M NaOH | Sigma-Aldrich | 72068 | |
100 nm carboxylated fluorescent polystyrene nanoparticles (FluoSpheres) | Invitrogen, (ThermoFischer Scientific) | F8803 |
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15 mL centrifuge tubes | ThermoFischer Scientific | 14-955-237 | |
20 G blunt needle | McMaster Carr | 75165A252 | |
25 mL tissue culture flasks | VWR | 10861-566 | |
3D printer | Lulzbot | LulzBot Mini 2 | |
3D printing software | Cura | Cura-Lulzbot | |
50 mL centrifuge tubes | ThermoFischer Scientific | 14-955-239 | |
Agar | Sigma-Aldrich | A1296 | |
Carbomer Granular Hydrogel Particles | Lubrizol | Carbopol 980NF | dry granules of crosslinked acrylic acid/alkyl acrylate copolymers |
Centrifuge (2 mL tube capacity) | VWR | 2405-37 | |
Centrifuge (50 mL tube capacity) | ThermoFischer Scientific | 75007200 | Sorvall (brand) ST 8 (model) |
Confocal Microscope | Nikon | A1R+ inverted laserscanning confocal microscope |
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Glass bottom petri dish | Cellvis | D35-10-1-N | |
Lennox LB (Lubria Broth) | Sigma-Aldrich | L3022 | |
M8 × 1.25 mm, 150 mm long, Fully Threaded Socket Cap | McMaster Carr | 91290A478 | |
M8 × 1.25 mm, Brass Thin Hex Nut | McMaster Carr | 93187A300 | |
Open-source syringe pump | Custom-made | Replistruder 4 | https://www.sciencedirect.com/science/article/pii/S2468067220300791 |
Petri dish (60 mm round) | ThermoFischer Scientific | FB0875713A | |
Shear Rheometer | Anton Paar | MCR 501 | |
Ultrasonic cleaner | VWR | 97043-992 |