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Harvard Researchers Snag $300K NIH Grant to Develop Single-Cell Array to Study Bacteria

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The National Institutes of Health has awarded a team of Harvard University researchers nearly $300,000 to develop a new microarray platform capable of imaging millions of bacterial cells in an overnight experiment.

Such a device could enable scientists to "probe the growth dynamics and the expression dynamics of very rare bacterial phenotypes" from cell to cell, according to Jeffrey Moffitt, a postdoc in the department of chemistry and chemical biology at Harvard.

Moffitt helped write the grant application with principal investigator Philippe Cluzel, a professor of molecular and cellular biology at the university whose research is focused on Escherichia coli and other bacteria. The NIH awarded the team the grant earlier this month and it is set to expire on June 30, 2014. The amount budgeted for the first year of the grant is $298,510.

Moffitt told BioArray News this week that the team was moved to develop a high-throughput platform for single-cell analysis because of shortcomings in conventional cell-screening methods.

"When you study bacterial dynamics at the single-cell level, you have a lot of problems associated with controlling the environment," Moffitt said. He noted that bacteria can spread, crowding the surface of the substrate, and making it difficult to conduct image analysis, as it is unclear if the bacteria are staying in the same chemical environment over time.

"We were interested in creating structured environments where we could control the bacterial density and we could control the way we deliver nutrients to them, so that we knew the chemical environment was constant, and that everything was set up so that you could do very easy image analysis after the fact," he said.

The group had already made progress toward that goal prior to receiving NIH support. It published a paper in Lab on a Chip earlier this year that described the patterning of cells on agarose gel pads on the sub-micron scale in linear tracks that "constrain the growth of bacteria into a high-density array of linear micro-colonies."

According to that paper, buffer is flowed through the microfluidic lines, washing away the excess cells, and delivering fresh nutrients. The researchers claimed they were able to cultivate and image hundreds of thousands of cells on a single agarose pad over between 30 and 40 generations. As a proof of principle, they looked at a community of Escherichia coli auxotrophs that can complement the amino acid deficiencies of one another.

Moffitt said that with the new funding, the team will continue its construction of the device, which it refers to as a single-cell chemostat, because it provides a chemically static environment. It will also develop methods to leverage existing microarray technology to print thousands of distinct bacterial strains onto a single patterned hydrogel.

Moffitt said that the team is pursuing two approaches for printing cells to patterned pads, using Digilab's CellJet cell printer. In the first, it will print directly onto the agarose pads. In the second, it will use the arrayer to print to a microscope slide, and then will use contact printing to transfer cells on the slide to its device. "We have proof-of-principle experiments to validate both approaches, but since each approach has its own pros and cons, we are pursuing these two complementary methods to determine which is ultimately more reliable," said Moffitt.

Finally, the team is developing image-based techniques to perform high-speed, high-throughput, time-lapse microscopy of growth on a nano-patterned pad, and plans to develop the automated image analysis software needed to extract the "full history of growth, division, and fluorescent gene expression within cellular lineages from these large data sets," according to the grant's abstract.

Applications

Moffitt said the team's longer-term goal is to get the high-throughput cellular chip "up and running" so that it can screen a large number of bacteria and "follow the single-cell dynamics, how cells are growing in time, how they are expressing proteins, and how they pass it on to daughter cells in hundreds of thousands of cells in parallel."

He said that one "interesting application" of the device is its ability to probe the growth dynamics and the expression dynamics of very rare bacterial phenotypes. Another is to look at variation in gene regulation over time.

"There are great techniques for assaying gene expression [using] microarrays, high-throughput sequencing, high-throuput mass [spectrometry], but you don't capture any of the variation between cells with those technologies," said Moffitt. "You see some average protein expression, [but] you don't know if some cells express a lot, some express none, or they all express the average amount," he said. "Moreover, what you really need to do is look at gene expression or transcriptional activity at the single-cell level, where you can see this variation from cell to cell. You can see [how] the transcriptional behavior varies in time, as one cell passes on its transcriptional profile to daughter cells."

As an example, Moffitt referred to Cluzel's E. coli research. "Imagine taking a library of all the genes in E. coli, with one strain for each protein, and then patterning that library on this chip. You would have a reporter on the transcriptional activity of every single operon that you could measure simultaneously in massive throughput," Moffitt said.

He said the ability to look at how every gene in E. coli behaves in living cells "unprecedented" and the ultimate goal of the device's developers. "You can look at the transcriptional behavior of the entire genome in real time in single cells," Moffitt said. "That [could be] the most exciting and novel dataset to come out of this device."

In terms of making the technology more widely available, Moffitt said that it would be "very easy" to disseminate the device to other labs working on other kinds of bacteria, and that Cluzel's lab is open to collaborations. "There is always the possibility of commercialization down the road, but that's not something we've talked about," he added.

Other Platforms

The Harvard team is one of a number of groups developing next-generation cell microarray platforms. In the past year, at least three other teams have discussed similar projects.

Last year, the NIH awarded $1.5 million to a team of researchers at Arizona State University to develop a high-throughput, live cell array for metabolic studies called Cellarium that the researchers claimed will enable users to perform "dynamic multiparameter measurements" on live cells in a high-throughput fashion.

The ASU researchers said at the time that they hope to use the chip to measure "key parameters in cell metabolism" such as oxygen consumption, pH, adenosine triphosphate, and glucose, in order to gain insight into how these parameters change over time, yielding information about cell life and death processes, neoplastic progression, and pyroptosis or proinflammatory cell death (BAN 12/13/2012).

Other research teams building high-throughput cell arrays include a group at the Broad Institute that has developed an array that relies on lentiviruses to deliver RNAi or open reading frames into cells (BAN 5/12/2012); a group at the VTT Technical Research Centre in Finland that has developed a platform that it claims can be used in large-scale gene knockdown analyses (BAN 4/12/2011); and a team at McGill University in Montreal that has designed another "living microarray" to study transcriptional changes in real time in single mammalian cells (BAN 2/22/2011).

And such activity isn't restricted to academia. For instance, Biolog, a Hayward, Calif.-based company, sells microarrays for cell phenotyping.

"Several groups are creating living analogs of microarrays, where the arrays are living cells, and it looks like a lot of people have been doing that for eukaryotic cell lines," said Moffitt of these other efforts. "We are hoping to essentially do that for bacteria."

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