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Web Date: April 26, 2012

Uncovering Cellular Contamination

Assay Development: Glowing enzyme detects mycoplasma hiding in cell culture
Department: Science & Technology | Collection: Life Sciences
News Channels: Analytical SCENE, Biological SCENE
Keywords: mycoplasmas, cell culture, contamination, luciferase
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Tiny But Terrible
Mycoplasma colonies grow on agar, magnified 10 times.
Credit: Lesley Young, U.K. Stem Cell Bank
Micograph of mycoplasma cells
 
Tiny But Terrible
Mycoplasma colonies grow on agar, magnified 10 times.
Credit: Lesley Young, U.K. Stem Cell Bank

Many microbes can contaminate mammalian cell cultures, but some of the most worrisome are mycoplasmas, members of a family of tiny bacteria that threaten cell viability and alter cell behavior. Now something good has come out of a set of accidentally infected cultures: Researchers have developed an analytical method that uses a glowing enzyme to quickly assess whether mycoplasmas have invaded (Anal. Chem., DOI: 10.1021/ac2033112).

Researchers have estimated that between 15 and 70% of mammalian cell cultures suffer from mycoplasma contamination, often caused by cross-contamination or nonsterile practices. Because mycoplasmas can alter a number of cell processes—including gene expression, cell metabolism, and cell proliferation—experiments unknowingly done on infected cell lines can lead to misleading data.

Once a mycoplasma infection is found, however, all is not lost. Labs can eliminate mycoplasmas with special antibiotics active against them, such as Plasmocin, without detriment to the cell cultures, says neuroscientist Bakhos Tannous of Harvard Medical School.

Currently, the most reliable way to detect mycoplasmas involves extracting and amplifying their DNA with polymerase chain reaction (PCR) techniques. “But PCR is time consuming, and you can have false positives and false negatives,” Tannous says.

He and his colleagues developed a faster technique serendipitously after noticing unusual behavior in a biological assay they were using to study brain tumor cells. They had genetically engineered Gaussia luciferase, a luminescent protein from a small marine crustacean, into the cells to monitor gene expression. In the assay, tumor cells synthesize the protein and then secrete it into the cell medium, where the researchers can sample it.

But when the researchers performed the assay on several cell lines, they found that the half-life of the luminescence varied greatly. “Sometimes it was seven days, which was expected; sometimes it wasn’t even 24 hours,” Tannous says.

Around this time, Tannous happened to ask his lab members whether they had performed their routine screening for mycoplasmas that month. They hadn’t. When they tested the cell cultures, they discovered that only contaminated ones showed the drop in luminescence.

To investigate further, they infected other brain tumor cells with three common species of mycoplasma and measured Gaussia luciferase luminescence. After three days, the contaminated cultures showed a 14 to 81% decline in the protein. They also ran the test with only mycoplasmas and no mammalian cells and found the same decrease in Gaussia luciferase.

Their method also could detect low concentrations of the bacteria that commercial tests could not. The team hypothesizes that mycoplasmas produce a protease that breaks down the luciferase protein.

Tannous thinks the technique could be of use to any laboratory employing mammalian cells. “It’s a very simple assay,” he says. Since a lot of cells are already engineered to express Gaussia luciferase, monitoring those cultures would be easy. Researchers could also add the protein to the cell medium, he says, instead of to the cells.

“This looks like a very interesting new test,” says Charles Rosser, of M.D. Anderson Cancer Center Orlando. “It’s very important to know if these cells are infected.” In earlier work, he and his colleagues found that mycoplasma infection caused benign prostate cells to look cancerous.

Rosser says the new technique could make it easier for a lab to screen many cultures at once: “To do 30 cultures the old way would take a substantial amount of time. To do 30 cultures this way, you could do it all in one morning and have the results by lunchtime.”

 
Chemical & Engineering News
ISSN 0009-2347
Copyright © American Chemical Society

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