Cell Culture Troubleshooting

Solutions for common problems with cell culture
Cell culture has become one of the most fundamental techniques for modeling biological systems.

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.

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Cell culture has become one of the most fundamental techniques for modeling biological systems.

Cell Line Misidentification

Extensive genetic testing by multiple cell repositories and other life science entities over more than two decades has revealed that cancer cell lines commonly used in research may be misidentified or cross-contaminated with other cell lines. 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: in the 1960s, isoenzyme analysis of multiple independent cell lines demonstrated that many shared a rare enzyme isoform with HeLa cells. HeLa has since been shown to be the most common invasive cell line in culture stocks
  • Substandard technique: Even the most conscientious cell culturist may expose cell stocks to cross-contamination during handling. Training and monitoring of all lab personnel in sterile culture technique along with single-use pipette tips and serological pipettes, sterile filtration of media, supplements, and buffers, and disinfection of biosafety cabinets and cell culture surfaces are key to minimizing corruption with opportunistic cell lines.
  • 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.

Microbial contamination of cell cultures

Cell culture media and incubation conditions provide the ideal environment for cells, as well as bacterial, fungal, and viral contaminants. In addition to diligent adherence to aseptic culture technique, cultures must be regularly examined microscopically for evidence of bacterial and fungal intruders. Some microbial contaminants, however, evade visual detection.

According to studies by the US FDA, ATCC, and others, it’s estimated that up to 30% of all cultures are contaminated with mycoplasma. Reagents and kits designed for detection of mycoplasma are often based on PCR amplification, which can also be used to detect viral contaminants.

Poor cell growth, detachment, clumping, and cell death in culture

When cells die in culture or fail to attain confluency, adherent cells spontaneously detach, or cells clump in suspension, contamination with microorganisms must first be ruled out. Other factors that may cause poor cell health or behavior inconsistent with phenotype include

  • Frozen stock conditions
  • Cell passage number/overconfluency
  • Environmental stress
  • Culture/freezing media, supplement quality
  • Overdigestion with dissociation enzymes
  • Inaccurate cell enumeration during freezedown or passage

Appropriate equipment, reagents, and protocols are key to resolving unexpected cell culture outcomes.