Controlling Cell Culture Contamination

 Controlling Cell Culture Contamination: Practical Steps

Cell culture contamination can be minimized through effective management practices. Laboratories, especially larger ones, can face significant issues if basic procedures aren't followed. By actively managing your cell cultures, you can address and reduce contamination risks. Setting up a culture management program tailored to your lab's needs and past experiences with contamination can help. This program can be informal or more structured based on your specific requirements.

Step 1: Understand the Problem

The first step in managing contamination is identifying the extent of culture loss in the lab. Everyone should keep detailed records for at least a month, noting all issues that lead to culture loss. These problems can range from contamination to equipment failures. After documenting the problems, the next step is to review them collectively. This helps to identify patterns and determine how frequent and serious the contamination is. Understanding the nature of these losses is crucial in deciding if a management system is necessary. Once the scope of the issue is understood, problem-solving techniques can be used to address the causes and prevent future issues.

Step 2: Implement Good Aseptic Techniques

Aseptic techniques are fundamental to preventing contamination. These techniques act as a barrier between microorganisms and cultures while allowing lab workers to handle the cultures safely. Training all laboratory personnel in proper aseptic practices is essential. One important recommendation is to use sealed culture vessels whenever possible, especially for long-term cultures. Additionally, use vented cap flasks, as these reduce contamination by allowing sterile gas exchange while keeping out microorganisms.

Step 3: Reduce Accidents

Accidents in the lab often lead to contamination, and they can be more common during stressful times, such as before holidays or when new staff members are working. To prevent accidents, ensure that all solutions and cultures are clearly labeled. A color-coding system can help reduce confusion in a busy lab. Always use standardized record-keeping forms and written protocols for media and solution preparation. These steps help minimize errors and allow for easier identification of issues if they arise.

Step 4: Maintain a Clean Work Area

A clean lab environment is crucial for preventing contamination. Regular cleaning of floors and work surfaces helps minimize airborne particles. Pay special attention to incubators and laminar flow hoods, as they can accumulate contaminants. Water baths used for thawing sera and warming media should be cleaned regularly, as they can harbor bacteria and other microorganisms. Waste disposal areas should also be kept clean, as improperly disposed materials can introduce contaminants. Lastly, regular maintenance of refrigerators and freezers can prevent microbial buildup and improve their efficiency.

Step 5: Additional Considerations

For some labs, pest management programs may be necessary to address contamination risks from rodents or insects. Potted plants, which can harbor pests, should be avoided near culture areas. Additionally, if pesticides are used, take care to prevent chemical contamination of cultures. By following these strategies, labs can significantly reduce contamination risks, leading to more reliable and productive cell culture work.

By using good aseptic techniques, preventing accidents, maintaining a clean environment, and taking additional preventive measures, labs can minimize contamination and ensure the success of their cell cultures.

Sterility Testing in Cell Cultures

Sterility testing is essential to prevent contamination in critical experiments. To ensure successful contamination control, it's important to monitor supplies, media, solutions, work areas, and cell cultures regularly. Keeping the testing process simple and effective encourages consistent use. Since no single method can detect all biological contaminants, testing often misses low levels of contamination, especially when antibiotics are present. However, several practical methods for monitoring contamination can be adapted to most cell culture labs.

Maintaining Equipment for Sterilization

Autoclaves and dry heat ovens, used to sterilize supplies, must be properly maintained, and staff should be well-trained in their operation. Thermometers and chart recorders used in sterilization should be calibrated regularly to maintain accuracy. Spore test strips or thermometers can be placed inside the autoclave to verify proper function. Additionally, all in-house filter-sterilized solutions should be tested for sterility each time before use.

Testing for Contaminants in Cell Culture Media

Cell culture media, especially outdated bottles without antibiotics, can serve as an effective substitute for standard microbiological media. A small amount of serum can be added to promote growth. Sterility testing involves placing a small sample of the medium in separate tubes, incubating at 30°C and 37°C for two weeks. This test can be helpful for identifying contaminants and evaluating potential sources of contamination, such as equipment or aseptic technique.

Mycoplasma Detection

Mycoplasma contamination in cell cultures can be difficult to detect. Direct culture in media is the most effective method, though it’s time-consuming and may take up to 28 days to yield results. Indirect testing methods, such as PCR kits and DNA fluorochrome staining, are faster and can detect even the most difficult strains, but they are less sensitive and may produce false negatives. A combination of both direct culture and DNA staining offers the most reliable results. Mycoplasma detection is especially important for newly acquired cell lines, which should be quarantined and tested for contamination.

Detecting Other Biological Contaminants

For detecting bacteria, yeast, and fungi, standard microbiological media can be used for culture tests. Indirect tests for mycoplasma can also identify these other contaminants. Protozoan contaminants may require special culture procedures. Additionally, continuous monitoring of cell cultures for genetic alterations, mutations, and chromosomal changes is essential. Methods such as karyotyping, electrophoresis, and DNA fingerprinting can help track these changes and ensure that cell lines remain reliable for research.

Recommendations for a Testing Program

A robust testing program is the foundation of reliable research. Initially, test all in-house cell lines for mycoplasma and microbial contaminants, and ensure they are properly identified. Quarantine and test all incoming cell lines, and periodically test cell lines in continuous use, especially when they show unusual behavior. New lots of sera should also be tested before use in critical applications. Regular testing helps maintain the integrity of cultures and prevents the spread of contamination.

Detecting Chemical Contaminants

Chemical contamination can alter the growth and behavior of cell cultures, often leading to misdiagnoses of biological contamination. Monitoring changes in the lab environment, such as new reagents or media, can help identify the source of the problem. Testing new lots of reagents, media, and sera, as well as evaluating water purity, can prevent chemical contamination. Regular record-keeping is crucial to identify possible causes of contamination.

Using a Frozen Cell Repository

A cryogenic cell repository helps maintain a backup of tested, contamination-free cell cultures. This approach reduces the need for extensive quality control testing and helps replace cultures lost due to contamination. By regularly discarding old cultures and replacing them with tested stocks from the repository, labs can avoid potential contamination and reduce testing efforts. For efficient use, stock cultures should be grown in antibiotic-free media, thoroughly tested, and frozen in batches that will last throughout a research project. This method ensures that only newly introduced cultures are tested, limiting testing to practical levels.

By following these guidelines, laboratories can ensure the reliability of their cell cultures and protect the integrity of their research from contamination.

Strategic Use of Antibiotics in Cell Culture

Antibiotics can be a helpful tool in cell culture when used wisely, but they can be harmful if overused or used incorrectly. Experts have long recommended that antibiotics should not be used routinely in culture media. A study by Barile found that 72% of cultures grown with antibiotics were contaminated by mycoplasma, compared to only 7% of cultures grown without antibiotics, highlighting the dangers of constant antibiotic use. Overreliance on antibiotics often leads to poor aseptic technique and antibiotic resistance, particularly among common culture contaminants. In a study of mycoplasmas, most strains were found to be resistant to commonly used antibiotics, including gentamycin, erythromycin, and kanamycin.

Antibiotics can cause more harm than good because they often allow undetected contaminants, such as mycoplasmas, to grow while preventing the growth of visible contaminants. This results in cultures being kept in use even though they are infected. As a result, antibiotics should only be used strategically and for short periods, such as during the early stages of primary cultures or hybridoma production. When using antibiotics, it’s essential to choose ones that are effective, non-toxic, and stable.

Dealing with Contaminated Cultures

Autoclaving is the most reliable method for handling contaminated cultures, as it prevents infection from spreading. However, in some cases, valuable cultures may become contaminated and efforts to save them may be necessary. This process can be challenging and often unsuccessful. Fungal or yeast contamination, for instance, is difficult to treat since antifungal agents only prevent growth, not kill the organisms. Mycoplasma contamination is typically the focus of cleanup efforts, but many strains of mycoplasma are resistant to common antibiotics used for this purpose. Repeated attempts to clean cultures may lead to antibiotic resistance in mycoplasma strains, making future cleanup efforts even harder.

Other methods, such as combining antibiotics with specific antisera or chemicals, are sometimes used, but none are foolproof. Mycoplasma detection often becomes harder after treatment because low-level contamination may go undetected. While some commercial mycoplasma cleanup services exist, they can be expensive and are not always successful.

The High Cost of Contamination

Cell culture contamination, especially by mycoplasma, costs researchers millions of dollars annually, as it disrupts experiments and leads to the loss of valuable cultures. Unfortunately, contamination remains a widespread problem, with many cell culture workers facing issues with mycoplasma. A survey showed that 23% of respondents had experienced mycoplasma contamination, while 44% suspected it but were unsure. Many researchers do not test for mycoplasma, with 50% of survey respondents admitting they don’t check for it in their cultures. The overuse of antibiotics is a significant factor contributing to this problem.

Preventing Mycoplasma Contamination

To reduce the risk of mycoplasma contamination, it's essential to identify its main sources. The most common source is infected cell lines, so all new cultures should be quarantined and tested for mycoplasma before being used in research. Another source is poor aseptic technique by the cell culturist, which can be minimized by using tested cell lines and limiting antibiotic use. Sera and other biological materials can also introduce mycoplasma if not properly sterilized. Always purchase these materials from reputable sources that use adequate filtration and testing methods.

While it is impossible to completely eliminate contamination, understanding its causes and taking preventive measures can significantly reduce its impact. Developing a contamination management plan based on these principles can help keep your lab cultures healthy and your research on track.