Types of Cell Culture Contamination and How To Prevent Them
Avoiding contamination is essential for successful cell culture.
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What is cell culture?
Cell culture, or tissue culture, refers to the growth and maintenance of living cells in vitro – outside a living organism. Since Ross Granville Harrison first sustained frog nerve fibers in test tubes in 1906, cell culture has become an essential technique in life science research.1 Over the last century, it has played a part in some enormous leaps in scientific knowledge and understanding, including the development of induced pluripotent stem cells, gene editing and vaccine development.2
Today, cell culture’s wide-ranging applications include genetic engineering, drug development and vaccine production. Although the foundational concepts and methods remain the same, the technology has advanced far beyond test tubes full of blood and agar. Emerging techniques, such as 3D cell culture models and organ-on-a-chip, emulate complex multicellular organs and cellular interactions, enabling the simulation of human physiology as closely as possible in both normal development and diseased states.
However, even as the core concepts of cell culture remain the same, so too does the main roadblock to successful cell culture: contamination. Unfortunately for cell culture scientists, the conditions in which cells thrive (nutrient-rich media, oxygen, carbon dioxide) are also the same conditions in which many contaminants thrive. While in vivo cells are protected by the immune system, there is no immune response in a tissue culture flask, so the cells are vulnerable to opportunistic pathogens and contaminants. Contamination can have significant effects on cells, including slowing cell growth, altering morphology and increasing death rates, all of which can have knock-on effects on any experiments performed and data collected. Here, we’ll discuss the different types of cell culture contamination, how to identify and treat contamination as soon as possible and how to prevent it entirely, ensuring you can keep your cells healthy and happy.
Types of cell culture contamination
- Mycoplasma contamination
- Bacterial contamination
- Fungal contamination
- Yeast contamination
- Mold contamination
- Viral contamination
- Other types of contamination
How to identify contamination in cell culture
How to get rid of cell culture contamination
Types of cell culture contamination
Several different types of microbes can contaminate cell culture, including bacteria, fungi, viruses and Mycoplasma. Some of these, such as mold contamination, can be obvious, and therefore caught quickly, while others, such as Mycoplasma, can fly under the radar for long periods. Cell lines can also be contaminated with other cell lines, which can – over time – outcompete and take over the original cells. Understanding each type of potential contamination is key to removing contaminants and preventing future unwanted intrusions.
Mycoplasma contamination
Mycoplasma are one of the most common contaminants in cell culture labs and one of the hardest to spot, as they are not visible under a standard light microscope. Mycoplasma are tiny prokaryotes that lack a cell wall and express only a very small genome (approximately 800 kbp, compared to an average 5 mbp for bacteria).3 Despite their differences, Mycoplasma species are thought to have degeneratively evolved from gram-positive bacteria and have retained the ability to replicate independently of other cells.
The most common species of Mycoplasma found in cell culture are Mycoplasma fermentans, Mycoplasma orale and Mycoplasma arginine. The origins of contamination are typically reagents of animal origin and operator cross-contamination – or even the human operators themselves. Mycoplasma contamination is an enormous issue in tissue culture, as expansive screening has suggested that 15–35% of all continuous cell lines are infected with Mycoplasma, with the common movement of cell lines between labs providing ample opportunity to spread.4
Although Mycoplasma contamination doesn’t produce any visible or morphological symptoms in infected cultures, it can have profound effects on cell functions, including changes in gene expression, protein synthesis and secretion and metabolism (Figure 1).4 In turn, this can significantly affect the quality and reliability of data produced from experiments using contaminated cells – even changing the response of cancer cell lines to chemotherapy.5,6
Figure 1. The major impacts Mycoplasma contamination can have on cellular functions in cell cultures. Credit: Technology Networks.
Bacterial contamination
Fungal contamination
Yeast contamination
Mold contamination
Viral contamination
Unlike other types of contamination, which typically occur from environmental or operator sources, viral contamination can often originate in the cell line itself. For example, in the 1950s and ’60s, kidney cells isolated from macaques for use in the polio vaccine were later discovered to be contaminated with simian virus 40 (SV40).8 In some cases, contamination with human or zoonotic viruses such as human immunodeficiency virus (HIV-1) and lymphocytic choriomeningitis virus (LCMV) can put scientists at risk of infection and require a higher biological safety level than uncontaminated cells.7
Other types of contamination
| Contaminant | Examples and sources | Detection | Removal or prevention |
| Parasites | Intracellular protozoan parasites may be found in primary cultures, including Toxoplasma gondii, Cryptosporidium parvum and Plasmodium species. | Visual detection of intracellular parasites will vary according to the parasite in question, but suspected contamination can be confirmed with testing, e.g., PCR. | High-risk primary cell cultures should be tested for parasitic contamination before use, and appropriate containment methods should be used when working with such cell lines to avoid laboratory-acquired infections. |
| Prions | Some cell lines can be susceptible to prion infection, which can originate from fetal bovine serum (FBS) supplements. | Prions cannot be detected by visually examining culture, but at-risk reagents should be tested. | Use high-quality media supplements with low risk for prions, and test reagent batches regularly. |
| Chemical | Includes detergents, hormones, heavy metals and plasticizers, introduced from plasticware or reagents. Endotoxins can be introduced from cell culture media and supplements. Free radicals can be induced by certain culture conditions, e.g., high fluorescent light exposure. | Difficult to observe, but can inhibit growth and replication. | Ensure water and reagents are tested for heavy metal contamination. Only use media and supplements from reliable sources that have certified low endotoxin levels. Free radical scavengers such as vitamin A or E can be added to media. |
| Inter- and intra-species cross-contamination | Unrelated cell lines from the same species, or from another species. | Overgrowth of cells with unfamiliar or different morphology or unexpected characteristics. Regular testing can also detect contamination. | Any new cell lines must be characterized on entry to the lab, and existing cell lines should be tested regularly, as cross-contamination can result in unreliable and unreproducible findings. Testing can be performed by genetic sequencing or isoenzyme analysis. |
How to identify contamination in cell culture
| Contaminant | Sources | Detection | Removal or prevention |
| Mycoplasma | Reagents of animal origin and operator cross-contamination. | Changes in growth rates or morphology of cells, in conjunction with regular testing of cell lines. | Infected cell lines should be discarded, and the lab disinfected. Mycoplasma-specific antibiotics can be used for irreplaceable cells. All new cell lines entering the lab should be quarantined and tested using commercially available testing kits. |
| Bacteria | Poor aseptic technique, reagents warmed in contaminated water baths and operator cross-contamination. | Visual changes in media (e.g., color change or cloudiness) or changes in cell growth. Motile bacterial cells may be visible under the microscope. Contamination can be confirmed using microbiological culture techniques or PCR. | Infected cell lines should be discarded, and the lab disinfected. For irreplaceable cells, antibiotics can be used. Aseptic technique is the best method of prevention. |
| Fungi | Poor aseptic technique, reagents warmed in contaminated water baths and operator cross-contamination. | Molds are easily visually identified as furry patches in the media. Yeast contamination can cause media turbidity and may be visible microscopically as budding chains. | Infected cell lines should be discarded, and the lab disinfected. Antimycotics can be used for irreplaceable cells. Aseptic technique is the best method of prevention. |
| Viruses | Primary cell cultures, horizontal transmission from other cell lines. | Some cytopathic viruses may cause cell death, while other species are only identifiable by electron microscopy. Contamination can be confirmed with cell- or PCR-based assays. | Infected cell lines should be discarded, and the lab disinfected. Safety protocols should be assessed to prevent laboratory-acquired infections. New, high-risk cell lines should be quarantined and tested. |
How to get rid of cell culture contamination
The best course of action is to immediately dispose of any contaminated cultures and disinfect anything they may have come into contact with the contaminated culture, including water baths, incubators, laminar flow hoods or Class II cabinets. Depending on the source of the contamination, reagents may also need to be tested or disposed of, or staff may need to be retrained.
In some cases, however, disposing of a cell line may not be possible. If an irreplaceable cell line becomes contaminated, it is possible to try eliminating or controlling the offending contaminant – if it is bacterial, fungal or Mycoplasma.
- The first step is to identify the contaminant: is it bacterial, Mycoplasma, fungal or viral? The gold standard for Mycoplasma, bacteria and fungi is culturing, but this can take several days – too long when contamination needs to be controlled. Therefore, faster methods such as PCR, DNA staining or sequencing can be used. Commercial Mycoplasma testing kits are also readily available.
- Isolate the contaminated cultures and thoroughly clean the lab with appropriate disinfectants.
- Some treatments can be toxic to cells, so dose-response testing may be required. This can be performed by collecting the contaminated cell suspension and seeding it at a standard dilution into fresh plates or flasks before adding the appropriate antibiotic or antifungal in varying concentrations (Table 2). Cells should be observed for signs of toxicity (e.g., poor growth, sloughing and rounding) for several days.
- Once potential toxicity has been determined, the cell line can be cultured for two to three passages in media containing the appropriate concentration of antimicrobial.
- Culture the cells in an antibiotic-free medium for one passage and again in treated media for two to three passages.
- Culture the cells in an antibiotic-free medium for several passages to determine if the contamination has been removed. If it still shows signs of contamination, repeat steps 4–5.
When and how to use cell culture antibiotics
Antibiotics can be used as treatments to clear bacterial or Mycoplasma contaminations, but many labs also use them as a preventative measure in standard media. Cocktails of antibiotics such as penicillin and streptomycin are added to media before use to prevent bacterial contamination. Preemptive antibiotics can be useful in some situations (e.g., for primary cell cultures, critical experiments or selecting for genetically modified cells). However, when overused or misused, antibiotics can have adverse effects. The constant presence of antibiotics can create a false sense of security, leading to complacency in the implementation of good aseptic technique. One major study found that cell cultures grown with continuous antibiotics had ten-fold more Mycoplasma contamination than those grown without.9 Additionally, continuous use of antibiotics can lead to contamination with slow-growing antibiotic-resistant organisms that cause subtle changes to cell behavior but are difficult to identify or eliminate.
Prolonged use of antibiotics in cell culture can also affect the cells themselves. Studies have shown that antibiotic treatment with penicillin and streptomycin can affect gene expression, potentially altering cellular behaviors such as differentiation and protein dephosphorylation, while rifampin can induce genome-wide changes.10 The side effects of prolonged antibiotic use in cell culture should, therefore, be considered carefully, especially in sensitive studies where good aseptic technique can achieve the same result.
How to avoid cell culture contamination
While there are methods of treatment available for some forms of contamination, this can be time-consuming and call into question data collected from experiments previously performed with the same cell line. Highly contaminated cell lines may have to be disposed of to prevent the spread to other cell lines in the lab. In the case of contaminated stock cell lines, all infected stock may need to be destroyed. Therefore, prevention is certainly better than a cure in almost all cases.
Several avenues can be taken to prevent contamination, and these should be initiated as soon as a new cell line arrives in a lab. New cell lines are one of the most common sources of contamination, and all new cell lines should be quarantined in a separate lab or Class II cabinet and tested for common contaminants such as bacteria and Mycoplasma before they are introduced into the general lab. If possible, cell lines should also be tested to confirm their identity. Once a cell line is established in a lab, quality control in the form of routine contamination testing should be carried out at regular intervals. Any animal-derived reagents such as FBS should be treated before use, for example, with ultraviolet (UV) irradiation.
Many labs use cocktails of antibiotics such as penicillin and streptomycin as standard in their cell culture media, though – as discussed – these can have negative effects. Overall, the best method of preventing cell culture contamination is to have well-trained staff adhering to good aseptic technique in a clean, well-maintained laboratory.
The need for aseptic technique in cell culture has been apparent since the very early days of the method. Harrison’s research was challenged by repeated bacterial contaminations – until he adopted aseptic technique.11
Good aseptic technique and proper use of certified hoods and cabinets are essential for an efficient, contamination-free cell culture lab. Additionally, operators who are trained to spot contamination in the earliest stages can help contain the spread of any microorganisms by treating contaminations as early as possible. A comprehensive cleaning schedule can also be a significant factor in preventing contamination by reducing the number of environmental microbes in the lab. Laminar flow hoods and cabinets should be disinfected after every use, e.g., using ethanol and UV light. Water baths for warming media or defrosting reagents should be cleaned and disinfected regularly, and any spills or waste should be disinfected and disposed of appropriately. Any equipment entering the hood should be wiped down with ethanol or other appropriate disinfectants, and everything that the cell culture comes into direct contact with should be single-use and sterile.
With well-trained staff, a clean lab and routine contamination surveillance, contamination risk can be minimized, and a lab full of healthy, contamination-free cell culture can be maintained.
