The Francis Crick Institute in London opened its doors in 2016, bringing together dozens of biomedical research groups from different institutes. However, two years before the scientists arrived, the Crick was already concerned about keeping the new cell culture facilities free of contaminates. The Cell Services team, at that time part of Cancer Research UK’s Clare Hall Laboratory in Hertfordshire, began checking all the cell lines that people planned to bring to the institute.
As well as finding a number of misidentified cell lines, the Cell Services team encountered a lot that were contaminated with mycoplasma. Many of the research teams were surprised, according to the platform head, Ruth Peat. “For groups who never had such an extensive service before, it was a bit of an eye-opener.”
A lot of researchers are unaware of the contamination status of their cell lines, and the effect it may have on their experiments. But keeping cell cultures free of mycoplasma or other microbes can be made easier.
Sources of contamination
Mycoplasma is the collective name for a class of small bacteria. Mycoplasmas are common in cattle, and came to cell culture labs via fetal bovine serum and other media additives. But that was decades ago. All new serum is checked and contamination-free. “Nowadays, mycoplasmas are distributed from one culture to another,” says Cord Uphoff, senior scientist in the Department of Human and Animal Cell Lines at the Leibniz Institute DSMZ in Germany.
Cell cultures don’t spontaneously spread mycoplasma, or, indeed, any bacterial, fungal or other contaminants. “It's the people who are handling the cell cultures,” explains Uphoff’s colleague and department head, Hans Drexler. “If one cell culture contaminates another, it’s because people are not properly following good cell culture practice.”
Unfortunately, mycoplasmas are not easy to spot. “In contrast with bacteria or fungi contamination, where you easily see turbidity or cloudiness, you cannot see mycoplasma contamination with the naked eye,” says Alexandra Müller-Scholz, who develops microbiology detection products at pharmaceutical and laboratory equipment supplier and life-science services company Sartorius, based in Göttingen, Germany. Standard cell culture microscopes can’t easily visualize mycoplasmas, which can be as small as 0.1 µm. Contamination, says Müller-Scholz, “can be undetected for quite a while”.
Detecting a mycoplasma infection
Traditional tests for mycoplasmas involve growing cultures on agar plates, or using fluorescence microscopy to check for biochemical markers, but these can take days or weeks. A faster test is to use a PCR-based method, with primers specific to mycoplasma1.
Peat uses all three methods to test any cell lines coming to the Crick. If there is mycoplasma contamination but the initial PCR tests don’t pick it up, it will eventually show on the agar plate a few weeks later.
For researchers working with stem cells and other cell-based products intended for regenerative medical treatments, the traditional testing process is not ideal.
“They typically have products with a really short shelf life, so they cannot wait for growth-based mycoplasma detection tests,” says Müller-Scholz. To address the needs of this community, her team has developed high-sensitivity real-time PCR kits to check for various contaminations.
The overall incidence of mycoplasma contamination in leukemia-lymphoma cell lines has declined since 2000 (ref. 2). “Scientists are more aware of the problem,” explains Uphoff, “and there are many detection kits available.” Still, mycoplasmas persist. Uphoff and Drexler manage a facility at DSMZ where researchers can send their cells to be tested, and they have noticed an interesting correlation. “Cultures we get from universities, are very often contaminated with mycoplasmas,” says Uphoff, “whereas samples from companies are usually not.”
Academic labs have higher contamination rates because they often exchange cell cultures and they have a high turnover of cell-culture users. Until new users are fully trained in sterile techniques, they are a risk.
At the Crick, Peat organizes monthly cell culture training for researchers who are new to the institute or want a refresher. During the influx of new PhD students at the end of summer, training sessions increase to weekly until the entire new cohort is trained in sterile techniques and in the use of the Cell Services core facility.
Not all institutes have facilities that can carry out mycoplasma and sterility checks. For researchers who need to do their own tests, Müller-Scholz recommends that they start by doing a thorough risk assessment3. “What are the potential sources of bacteria, fungi, or mycoplasma contamination? Do you have many open handling steps? Where do your materials come from?” Guided by the risk assessment, researchers can determine a testing routine for their own cell culture lab.
Eliminating an infection
Once a bacterial or fungal infection is spotted, the entire cell culture environment and autoclaving equipment needs to be thoroughly cleaned, and culture media filtered or discarded, before experiments can resume using a clean source stock. With mycoplasma infections, however, this source stock might also be contaminated. In such cases, it’s possible to eliminate the mycoplasma using antibiotics, such as fluoroquinolones or tetracyclines4.
Uphoff and Drexler recommend avoiding the use of antibiotics in regular cell culture maintenance5. Antibiotics do not protect against all types of infection, and daily prophylactic antibiotic use can compromise the cells’ function6. It’s better to avoid contamination altogether by using sterile techniques, leaving antibiotics only for emergencies.
Antibiotic use has other issues. Peat has observed a need to use higher concentrations of antibiotics to kill the mycoplasmas. “The treatments we have are still working,” she says, “but there's definitely some resistance — and that will probably increase.”
Good cell culture practice
Contamination happens quickly, and can only be revealed after it occurs. The best defence is to maintain a clean cell culture environment and practise sterile techniques. Peat, Drexler and Uphoff all recommend several precautions to keep cultures clean and minimize the risk of cross contamination.
Good practice includes: changing pipette tips between aspirating different cultures; using autoclavable instruments; and having dedicated media bottles for each cell line. In addition, filters of different sizes can eliminate existing contaminants from media and water sources used in the cell culture environment3,7.
These methods will help reduce the risk of infection by any microbial contaminant. When it comes to mycoplasma, though, the main source of contamination is other cell cultures. Uphoff says his work at the Mycoplasma Detection Service at DSMZ proved this. “What we see is that cultures from one laboratory are either all contaminated with mycoplasma, or none of them are.” He sees this as further evidence that mycoplasma is transferred by the people who handle the cultures
With regular checks, a clean backup storage, and intensive training , the Crick is keeping ahead of contamination. And there is one golden rule that Peat insists on to keep it that way. “Only ever work on one cell line at a time.”