One of the most important methods for modelling biological systems nowadays is cell culture, which is also a vital step in life science research laboratories and is becoming more and more important in the biotechnology and pharmaceutical industries. The effective propagation of cells for stock expansion or modelling research might be hampered by contamination or other factors that have a detrimental influence on cell viability, despite the ease with which this approach can be used. Sharing cells often has resulted in widely reported proof of the contamination of cell pools whose identification was previously unquestionable. It is possible to improve lab productivity, boost the production of cellular products, and provide relevant, trustworthy downstream data from in vitro models by identifying both frequent and uncommon reasons why cells could not grow.
CELL LINE MISIDENTIFICATION
Recent studies estimate misidentification may affect up to one third of all cell lines in use. These discoveries have the potential to call into question published findings about biological systems based on results from cell line models. Factors leading to cell line misidentification/cross-contamination include:
- Contamination with aggressive cell lines: Isoenzyme analysis has demonstrated that many cell lines share a rare enzyme isoform with HeLa cells. HeLa has since been shown to be the most common invasive cell line in culture stocks.
- Documentation and labeling practices: Cell culture flasks, plates, cryovials, and freezer containment must be clearly labeled, and liquid nitrogen storage maps strictly maintained to reflect the addition, withdrawal, and relocation of cell stocks.
- Cell line borrowing: Sharing of cells amongst neighboring labs is a common practice that leads to errors in cell line identity. Cell lines should only be obtained from reputable cell repositories such as ECACC, the European Collection of Authenticated Cell Cultures at Public Health England.
Be informed about common causes of cell line cross-contamination and how to protect your work from its consequences.
MICROBIAL CONTAMINATION OF CELL CULTURES
Cells, as well as bacterial, fungal, and viral contamination, thrive under cell culture medium and incubation conditions. Cultures must be routinely inspected under a microscope for signs of bacterial and fungal intrusions to strengthen strict adherence to aseptic culture procedures. Since mycoplasma is thought to be present in up to 30% of all cultures, certain common microbial contaminants can go undetected by the naked eye. The PCR amplification method, which may be used to identify viral contamination, is frequently the foundation for reagents and kits intended for the detection of invisible mycoplasma.
Learn how to keep your cultures free of biological and chemical contaminants.
POOR CELL GROWTH
It’s frustrating when cells in culture that otherwise appear healthy fail to attain confluency. When contaminants are ruled out, some possible barriers to healthy cell doubling include:
- Culture/freezing media condition and quality
- Quality and application-suitability of supplements
- Inaccurate cell enumeration during freezedown or passage
When cells seem viable but fail to expand, learn what you can do to improve culture conditions for optimal growth before heading back to the liquid nitrogen.
DETACHMENT OF ADHERENT CELL PHENOTYPES
Adherent cells may spontaneously detach in culture, singly or in sheets. When adherent cultures don’t stay that way, know what to check and how to restore cohesion in culture.
Cells in suspension mimic in vivo conditions as singlets. When cells start to clump, this can be due to sticky nucleic acids, present in media when cultures are stressed. What’s hiding in media that can cause cells to clump? Read more here about how to banish clumping from cultures.
CELL DEATH IN CULTURE
When cells die in culture, contamination with microorganisms must first be ruled out. Other factors that may contribute to poor cell health or behavior inconsistent with phenotype include:
- Frozen stock conditions
- Cell passage number/overconfluency
- Environmental stress
- Overdigestion with dissociation enzymes
Appropriate equipment, qualified reagents, and expert protocols are key to resolving unexpected cell culture outcomes.
PRECIPITATES IN CULTURE MEDIA
If it’s not contamination, particulates in culture often have an inorganic explanation. Understand balanced buffer and media constituents, and learn more about keeping media constituents in suspension.