bioprinting bacterial colonies for motility and growth analysis in a porous hydrogel matrix

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

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.

Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

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

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.

Bioprinting Bacterial Colonies for Motility and Growth Analysis in a Porous Hydrogel Matrix

bioprinting-bacterial-colonies-for-motility-growth-analysis-porous

Research

JoVE Journal

Bioengineering

309 Views.  University of Colorado Boulder. Bioprinting Bacterial Colonies for Motility and Growth Analysis in a Porous Hydrogel Matrix

Video: Bioprinting Bacterial Colonies for Motility and Growth Analysis in a Porous Hydrogel Matrix

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.

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. ...

Methods Article

Bioprinting Bacterial Colonies for Motility and Growth Analysis in a Porous Hydrogel Matrix