Cell culture is expensive. But the cost of contamination of cell culture can be exorbitant. Sometimes the offenders are overt, causing media to turn opaque and milky or cells to suddenly become bespeckled, and you can cut your losses and start fresh. Other times, mycoplasma can run amok for months, invisible and odious, causing experiments to go haywire. A few years ago, researchers estimated that mycoplasma had the potential to affect hundreds of millions of dollars of NIH-funded research. And then there is contamination hiding in plain sight—misidentified cell lines, able to jeopardize years of experiments. (The most notorious may have been MDA-435, a “breast cancer” cell line that turned out to be melanoma-derived.)
Today, most journals require cell lines to be validated using STR (short-tandem repeat) or SNP (single nucleotide polymorphism) analyses for verifying cell-line identity. And many cell-line distributors assure authentication. But in the day to day, how can researchers protect cells (and future experiments) from the myriad offenders searching for a warm and humid place to call home?
You know the saying: An ounce of prevention is worth a pound of cure. This is especially true for working with cells, which must be inspected daily and tested for mycoplasma regularly. And lines should be validated prior to beginning any experiments (especially if they happen to come from the lab down the hall). Contamination will happen on occasion, but there are plenty of steps that can be taken to curb it.
Know your enemy
While mycoplasma detection and cell-line validation require some extra effort, simply looking at your cells under the microscope everyday will help to weed out other biological contaminants. According to Yana Li, laboratory manager of the eukaryotic tissue culture facility at the Johns Hopkins University School of Medicine, these include bacteria, molds, yeasts, and viruses. Li says that chemical contaminants such as impurities in the media, sera, endotoxins, and detergents can also be detrimental.
Use the medium as an indicator, paying attention to any rapid color changes (indicating a swift shift in pH, if phenol red is used) or increase in opacity. Familiarize yourself with what contaminated cultures look like and watch for bright particles in between cells (oftentimes yeast). Also, any changes in cell morphology—such as formation of cytoplasmic vacuolation, formation of nuclear granules, or reduced proliferation—are indicative of unhealthy cultures (and potential contamination).
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Learn how to identify potential contamination, steps to control mycoplasma contamination, tips for keeping a CO2 incubator contamination-free, how to clean a spill in a biosafety cabinet, what to do when contamination strikes, and three factors that contribute to a successful lab. “Always buy cell lines from reputable vendors who have characterized the cell lines and can certify that they are mycoplasma free,” advises Peggy Keefe, research assistant and lab manager for the University of Oklahoma Stephenson School of Biomedical Engineering. And be careful with gifts of cells from other labs. “They may be free at the onset but will cost you much later.” But, if you must use cells that come from another lab, quarantine them in a separate space until they’ve been tested and shown to be free from infection. Li says, “Before conducting experiments, those cells should be authenticated.”
Manage your workspace
“The specific requirements depend mainly on the type of research conducted. It’s preferable to maintain a separate tissue culture room to designate an aseptic work area for cell culture work,” advises Li. Ideally, cell culture rooms should also have separate ventilation and air conditioning systems. Equipment should be set up away from doors and other high-traffic areas.
“Do a good ‘footprint measure’ of the cell culture room to arrange your equipment. Limit the distance between equipment, let’s say the incubator where the cells are growing and the hood you are working in,” says Li.
Incubators should also be kept away from vibrations (such as from elevators or heavy equipment). If the space has windows, keep them closed at all times. Cell culture supplies should be kept close to where you are working to minimize air disruption.
Control your environment
Every surface and piece of equipment that comes into contact with cell plates must be thoroughly disinfected (70% ethanol or propanol works fine). This includes the microscope. Similarly, the biosafety cabinet (or flow hood) is meant to be a sterile sanctuary for cells. Ensure that the window sash is in the correct position, and wait several minutes until the airflow is established inside.
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Keefe says that the biggest mistakes scientists make include not waiting long enough for the hood to warm up and overcrowding the workspace inside the hood. “Only put in the hood what you will be using. Don’t use it for storage of pipet boxes, waste containers, etc.” This balance is critical because every single item and every movement that occurs inside of the hood can disrupt the laminar airflow and put cells at risk. When working, Keefe suggests using as much of the space as possible. “Move open bottles or flasks out of the way. You don’t want to pass your hands over top of them, increasing the chance of contamination.” And while having that bunsen burner on might feel like its offering some protection, in actuality it is also interfering with the airflow, so best to keep it off.
Zan Chen, research fellow in cell biology at Harvard University, says that oftentimes the vacuum system gets ignored and this is a mistake. “When the tubing gets old and [misshapen], the vacuum is no longer strong enough and sometimes the medium or buffer can backflush into cell culture dishes and cause contamination. It happened to us several times.”
Likewise, care should also be taken with the incubator. Although newer incubators are equipped with antimicrobial copper surfaces and decontamination settings, regular maintenance (like changing the HEPA filter and refilling the water pan) will keep things running smoothly. Try to minimize the time that the door is open and be careful to clean up any spills quickly and thoroughly.
Keefe says that her lab decontaminates the incubators more often since the introduction of auto decontamination cycles. “With the auto-decontamination cycle, the incubator is sterile overnight and back in service. Before, you had to move the cells, autoclave the racks, wipe it down with isopropyl alcohol, then reassemble the racks, add water, and wait for the humidity to go back up.” All of which took at least a day or two.
Li agrees that purchasing an incubator with a high temperature decontamination cycle is helpful. “Good equipment maintenance is essential for safe and efficient operations.” But she also thinks that a great management team is necessary: “Ensuring that equipment is properly maintained, supplies are readily available, and managing emergency cell handling during weekends and holidays is critical to keeping tissue cultures healthy.”
Check yourself
The most advanced antimicrobial equipment cannot save you from sloppy work or inexperience. This is because humans are covered in microbes. Particles from hair, skin, or clothing can blow into cultures, so precautions should be taken.
“Thorough training in tissue culture practices for anyone working in the tissue culture room will reduce the risk of contamination,” says Keefe. Researchers should change into a cell culture designated lab coat upon entry to the cell culture space (and consider shoe covers as well). Wash and disinfect your hands and then glove up and disinfect again.
Try to minimize talking, and if you must speak, turn your head away from cells. One of the predominant species of mycoplasma comes from human mouths, “especially when [they are] laughing, which can overwhelm the air barrier,” notes Keefe.
When contamination strikes
Despite best efforts, contamination will probably happen on occasion. Microbes are everywhere. But if cells are checked daily, mycoplasma is tested for regularly, and there are protocols in place for the maintenance and sanitizing of equipment, contamination can be downgraded from crisis to annoyance. When it does strike, there is little hope for the cells. Li says, “Once contaminated, most tissues or cells are only fit for the trash.”
Keefe says that on one occasion she’s witnessed primary cells brought back from a bacterial infection with antibiotics. But there was always a lingering doubt about how they had been changed. “It survived the treatment and tested negative for mycoplasma but the question if it had been altered because of the antibiotic treatment kept us from using it after the first experiment.”
The next step involves sleuthing and sanitizing. “After we find a contamination, we have to find the reason and troubleshoot the problem based on the type of contaminant. This is followed by a deep clean of the work area, checking the health of other cells, etc.,” explains Li.
In general, any infected cultures should be immediately removed from the space. Pour a 10% sodium hypochlorite (bleach) solution into the flask and let sit for a few hours to kill the offending microorganisms. All culture medium associated with or prepared at the same time as the contaminated cells should be tossed. The incubator and biosafety cabinets should also be thoroughly decontaminated with a diluted bleach solution. Note that this can be corrosive to metal, so rinse thoroughly and spray down with 70% ethanol or isopropanol.
Moving forward (but not without the basics)
Cell culture is moving at warp speed. With scientists switching from cell lines to primary cells and swapping two dimensions for three, attention to the details like basic aseptic technique has never been more important. By tweaking your cell culture set up, ensuring regular equipment maintenance and decontamination, and being more vigilant when in the hood, you can be sure your research continues on its trajectory, unencumbered by microbial hijackers.
If you want to learn more about avoiding contamination in the lab, download our free eBook “Strategies to Minimize Contamination in the Cell Culture Lab” and find out how to identify potential contamination, what to do when contamination strikes, and much more.
Hero image: Minimize talking in the lab when working with cell cultures, and avoid laughing as much as possible. One of the predominant species of mycoplasma comes from human mouths.