- Cell culture contamination is one of the most common and serious problems encountered in laboratories involved in biological research and biomanufacturing.
- Contamination can compromise the validity of experimental results, lead to the loss of valuable cell lines, and in industrial settings, result in significant financial losses. Understanding the sources, types, detection methods, and prevention strategies for contamination is essential for maintaining reliable and reproducible cell culture practices.
- Contaminants in cell culture can be broadly categorized into biological and chemical types.
- Biological contaminants include bacteria, fungi (yeasts and molds), mycoplasma, and viruses.
- Chemical contaminants encompass endotoxins, impurities in water or reagents, plasticizers from culture vessels, and residual disinfectants.
- Of these, biological contamination is more frequently encountered and often more immediately disruptive, though chemical contaminants can exert subtle, chronic effects on cell growth and gene expression.
- Bacterial and fungal contaminations are usually introduced through poor aseptic technique, contaminated reagents, or environmental exposure. These microbes often grow rapidly and can be detected visually—cloudy media, changes in pH (e.g., phenol red turning yellow), and turbidity are common signs. Fungal contamination, particularly from molds, may be visible as filamentous structures or colonies in the medium. Yeasts can also grow in clumps and change the appearance of the culture.
- Mycoplasma contamination represents a more insidious threat. These wall-less bacteria are extremely small, lack a rigid outer membrane, and do not cause visible turbidity. They are also resistant to many common antibiotics used in culture media. Mycoplasma can alter host cell metabolism, gene expression, and growth without noticeable signs, making them difficult to detect without specialized tests such as PCR, DNA staining (e.g., Hoechst), or ELISA-based assays. Studies suggest that a significant percentage of continuously cultured cell lines may be unknowingly contaminated with mycoplasma, which can invalidate experimental data.
- Viral contamination is less common in routine academic labs but can be of serious concern in biopharmaceutical production or primary cultures derived from human or animal tissues. Viruses can be introduced via contaminated serum, tissues, or cell lines, and often remain latent or undetected unless specific assays are performed. In research, retrovirus and adenovirus contaminations are sometimes detected when working with genetically modified cells or vectors.
- Chemical contamination, though less obvious, can stem from residual disinfectants, reagent impurities, or poor-quality plastics. These substances may not be detected easily but can affect cell viability, differentiation potential, and signal transduction. For example, endotoxins—lipopolysaccharides from Gram-negative bacteria—are potent inflammatory stimulants and can interfere with immunological assays even at very low concentrations.
- Preventing contamination requires strict adherence to aseptic technique and good laboratory practices. This includes working in properly maintained biological safety cabinets, using sterilized media and reagents, routinely disinfecting work surfaces, and minimizing the opening of flasks and plates. Regular screening of cell cultures for mycoplasma and other contaminants is strongly recommended, especially when working with valuable or irreplaceable lines. Using antibiotics can help control bacterial contamination temporarily but should not replace good technique, as it may mask ongoing contamination or promote antibiotic resistance.
- When contamination is detected, immediate action should be taken. For bacterial or fungal contamination, the affected cultures should typically be discarded, and the workspace thoroughly disinfected. In the case of mycoplasma, specialized treatments such as plasmocin or dedicated decontamination protocols may be attempted, though results are not always reliable. For critical or irreplaceable lines, re-derivation from frozen stocks or re-authentication is often necessary.
