Cell Culture Troubleshooting

Solutions for common problems with cell culture
Solutions for common problems with cell culture.

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.

Cell Line Misidentification

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.

Cell culture contamination

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. 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.

Learn how to keep your cultures free of biological and chemical contaminants.

Poor cell growth

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 cells in culture

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.

Cell clumping in culture

Cell Clumping

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 cell culture

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.

Cell culture precipitates

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.


Related Technical Articles

  • Detect mycoplasma contamination in cell cultures three ways: culture test, DNA stain test, or mycoplasma PCR test. Mycoplasma elimination kits rid cultures of contamination.
  • Common Cell Culture Problems: Poor Attachment of Adherent Cells -- Healthy and appropriately nourished cells re-engineer and optimize their attachment matrices. Cells that are cultured under suboptimal growth conditions attach poorly. When the culture surface provided is appropriate, the most common cause of failure of cells to attach to a substrate is environmental stress placed on the cells. Stress on cells in culture is mediated by biophysical conditions, or by components that are either present or formed in media.
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