Combatting Cell Culture Contamination

Cell culture contamination is among life science research's most common and disruptive issues, often resulting in lost time, unreliable data, and increased costs. Understanding the types of contamination and implementing proactive prevention strategies can significantly affect cell culture quality and research outcomes. This article will provide insights into lab practices that contribute to cell culture contamination and ways to address them.

Overlooked factors contributing to cell culture contamination

Routine lab practices, in combination with environmental factors, can result in cell culture contamination. “I’m always a little surprised to still see even experienced cell culturists making their culture media cocktail without filtering the mixture,” shares Robin Clark, Global Product Manager, Advanced Cell Culture Cell Biology Reagents and Tools at Millipore Sigma. “Even though bottled media, FBS, antibiotics and even supplements may be supplied with a sterility claim, it’s still necessary to filter the mixture once sterile products have been opened in the lab, even in the biosafety cabinet. In-house, we use the Stericup^® 0.22 micron filter device, which generally excludes undesirable microorganisms and particles without slowing filtration. It's critical to use a device with the right membrane for low protein retention, to make sure nutrient proteins in the base media and supplements make it through to the media cocktail.”

Seasonal variations also influence the likelihood of contamination. “Mold, a ubiquitous threat, can thrive on virtually any surface when moisture is present, further complicating contamination control,” explains Kris Wronski, Cell Culture Applications Scientist, Thermo Fisher Scientific. “Peak periods for airborne fungal spores vary by region and time of year. For instance, in Europe, the highest concentrations typically occur from late June to August, with occasional spikes in September. However, it is crucial to remain vigilant year-round, as fungal spores can be introduced into lab environments at any time via human activity and air conditioning systems.”

Leveraging technology to minimize cell culture contamination

“Biosensors that can detect cell layer changes may also alert researchers to problems with contamination without the need for repeated removal of cultures from the incubator. New automated image-based cell culture monitoring could reveal visible microbial contamination from bacteria and fungi but would not be useful for mycoplasma monitoring. However, automation could simplify and encourage regular mycoplasma screening by enabling automated collecting of supernatant for subsequent detection assays,” explains Clark.

GMP, Good Manufacturing Practices, plays a significant role in reducing cell culture contamination. “The adoption of automation, including robotic workflows and related technologies, represents a transformative advancement for cell culture operations. This is particularly significant in the development of cell therapies, where cells themselves become living medicines. In this context, contamination control becomes paramount, as any lapse can pose substantial risks to patient safety. Consequently, every component of the workflow—from lab equipment and cell processing devices to single-use technologies—must adhere to the stringent regulations of GMP. This rigorous standard applies not only to cell production but also to quality control activities involving cell culture assays and even R&D efforts focused on media screening,” shares Wronski.

The role of training

Comprehensive training highlights the different sources of contamination—such as microbial, viral, and chemical—and helps personnel recognize contamination signs early. This knowledge encourages proactive measures, such as timely cleaning, testing, and quarantining new cultures, and fosters a more vigilant lab culture overall.

“As aseptic technique is the most fundamental skill in any cell culture lab, published protocols, adequate training, and even regular recertification for personnel performing culture is best adhered to in both commercial and academic labs,” explains Clark.

Wronski explains why contamination issues were prevalent in the academic setting—“Transitioning from several years as a researcher in academia to a pharmaceutical GMP quality control environment was a truly eye-opening experience for me. Each individual had their own unique habits, despite some form of training in pipetting, aseptic techniques, and instrument use. In stark contrast, the GMP laboratory environment was highly structured and meticulous. Personal habits were not permitted; instead, comprehensive training programs were implemented, including a week-long pipetting course, several weeks of cell culture and aseptic technique training, and extensive training on SOPs for various instruments. These programs were designed to ensure repetitive performance of tasks with attention to every detail.”

Recent innovations in cell culture contamination prevention

“Mycoplasma can’t be detected by simple microscopic examination like other microbial contaminants, so the most sensitive and rapid methods for mycoplasma contamination detection are gaining traction,” Clark explains. “PCR remains the most practical standard for mycoplasma detection, although other isothermal molecular DNA-based amplification/detection without cyclers or gels may be on the rise as labs search for rapid screening without the need for specialty equipment. For PCR detection, new one-step PCR detection kits for mycoplasma can simplify and save time by offering premixed PCR reagents. Biosensor technology is intriguing for its potential to act as a surrogate for cell health using circuit impedance. For contaminants that impact cell health by changing monolayer or cell junction integrity, biosensors could alert scientists without the need to remove cells from incubation for manual examination. However, this may not be useful for mycoplasma contamination, as mycoplasma typically don’t kill—or detectably damage—cell layers.”

“One of my favorite innovations remains the active particle control system integrated into the Thermo Scientific™ Heracell™ Vios CR CO₂ Incubators,” shares Wronski. “These incubators are third-party certified for cleanroom compatibility and are suitable for ISO Class 5 and GMP grade A/B environments. Other features that contribute to cleanroom compatibility include CO₂ incubator ease of use, stainless steel exterior, electropolished interior and shelving, in-chamber HEPA filtration to further protect precious cultures, validated disinfection and cleaning procedures, including compatibility with the STERIS dry, non-condensing VHP^® process, and IP54 compliant electronics.”

Preventing cell culture contamination is essential for ensuring the reliability of experimental results and protecting valuable cell lines. By understanding the types of contamination and employing stringent preventive practices, researchers can significantly reduce the frequency and impact of contamination in cell culture. Regular training, vigilant monitoring, and a commitment to sterile techniques are the backbone of any successful contamination control strategy, helping maintain the lab's integrity and quality of cell cultures.