This image, created by reconstructing data from confocal fluorescence microscopy, shows Pseudomonas bacteria (green) clustered inside a gelatin "tire" (red). J. CONNELL ET AL., UNIVERSITY OF TEXAS AT AUSTINUsing a laser to activate cross-linking in a gelatin mold containing randomly scattered bacterial cells, researchers can “trap” the microbes in designated areas, dictating the 3-dimensional structure of the populations. The study, published today (October 7) in Proceedings of the National Academy of Sciences, could allow biologists to study the role of population architecture in cellular communication, while still allowing the flow of chemical messages.

“In microbial populations, there’s cooperation and there’s cheating and there’s competition, and so understanding how these very complicated things actually function is not something you can just do in a petri dish or a bulk broth,” said bioengineer Bryan Kaehr of Sandia National Laboratories in Albuquerque, NM. “In order for us to ever really understand...

The work comes from Kaehr’s graduate advisor, chemist and bioengineer Jason Shear at the University of Texas at Austin, who has been working in 3-D fabrication using biological materials for about 10 years. Shear and his colleagues had previously used the cross-linking technique—which uses a laser to activate a photosensitizer that promotes bond formation between the molecules of the mold—to build molecular “houses” of bovine serum albumin (BSA), into which they seeded bacteria that could swim into the various “rooms.” The researchers could then warm the houses to 37°C, causing the “doors” of the house to swell shut, keeping the bacteria in place.

“Although [the house is] physically restrictive, it’s chemically permissive,” said Shear. “These walls will transmit important biological signals, like quorum-sensing signals and antibiotics.”

But such a procedure is obviously limited to motile species of bacteria, and it left a lot to chance in terms of which bacteria ended up in which rooms of the house. Now, the researchers have tackled these problems by 3-D printing the molecular houses around the bacterial cells that are already embedded in a gelatin mold. The researchers simply cultured bacteria in liquid gelatin and then allowed the mixture to cool and solidify. “It’s basically Jell-O with things suspended in it,” said Shear. Then, based on where the bacteria settled during this process, the team designed a molecular house to segregate the bacteria as they wanted and subjected the gel to the cross-linking action of the 3-D-printing laser.

“This is the beauty of the technique—that it allows you to create any 3-D structure,” said engineer Aleksandr Ovsianikov of the Vienna University of Technology in Austria, who last month used a similar approach to grow human osteosarcoma cells in a 3-D mold, but was not involved in the present study. “So you have total freedom [of design].”

In a proof-of-concept experiment, the researchers examined the role of population structure in bacteria’s ability to resist an antibiotic. The team used the technology to nest a population of Staphylococcus aureus, a bacterium that is normally susceptible to β-lactam antibiotics, within a surrounding population of Pseudomonas aeruginosa, which produces an enzyme that defends against β-lactam antibiotics. The two bacteria are often found together in the human body—in chronic wounds, for example, or the lungs of patients with cystic fibrosis—and the researchers wanted to ask: “Could one bacterium actually protect the other?” said microbiologist Marvin Whiteley, a UT-Austin collaborator of Shear’s and an author on the paper. “We were able to show that you definitely could in regard to antibiotic sensitivity,” he said. And notably, it took just a few P. aeruginosa per picoliter to protect the inner S. aureus population from ampicillin, a β-lactam antibiotic.

Whiteley was excited by the results, but even more so by the technique, which he said could bring some much-needed quantitative measurements to microbiology. “Analytic chemistry is a great thing, and microbiology needs to use it more.”

Kaehr added that the technology could have applications in other areas. “Their work here focuses on microbial communities and bacteria, but this is really a problem in just understanding multicellularity,” he said. “This is a technique [that] will hopefully reveal new biology that you otherwise couldn’t understand.”

Furthermore, Ovsianikov noted, the system can be adapted to do more than simply trap cells within a structured environment. The same laser setup can be used to immobilize molecules at precise locations within the environment, to provide adhesion sites, or to carve out channels in the gel, rather than build walls. “This is a tool which potentially allows you to cross-link your gel, dress up your gel with biomolecules, or create channels in the same way,” said Ovsianikov. “This is a tool which is much more than 3-D printing.”

J.L. Connell et al., “3D printing of microscopic bacterial communities,” PNAS, doi:10.1073/pnas.1309729110, 2013.

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