Cell culture has become one of the most fundamental techniques for modeling biological systems, and is of increasing importance in the biotechnology and pharmaceutical sectors as well as an essential process in life science research labs. Though this technique is highly accessible, successful propagation of cells for stock expansion or modeling experiments can be plagued by contamination or other conditions that negatively impact cell viability. The common practice of sharing cells has led to well-published evidence of cross-contamination of cell stocks whose identity was previously unquestioned. Identifying both common and infrequent reasons why cells may fail to thrive can increase lab efficiency, enhance yield of cellular products, and ensure meaningful, reliable downstream data from in vitro models.
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:
Be informed about common causes of cell line cross-contamination and how to protect your work from its consequences.
Cell culture media and incubation conditions provide the ideal environment for cells, as well as bacterial, fungal, and viral contaminants. To augment diligent adherence to aseptic culture technique, cultures must be regularly examined microscopically for evidence of bacterial and fungal intruders. Some ubiquitous microbial contaminants evade visual detection, as up to 30% of all cultures are estimated to be contaminated with mycoplasma. Reagents and kits designed for detection of invisible mycoplasma are often based on PCR amplification, which can also be used to detect viral contaminants.
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:
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.
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.
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:
Appropriate equipment, qualified reagents, and expert protocols are key to resolving unexpected cell culture outcomes.
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.