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Tips To Be Very Particular Of Good Laboratory Practices For Primary Culture

While most lab reports are filled with “breaking” results there are few articles that bring out the importance of correct identification of cells and primary culture of cells. This becomes important given that the issue of “mistaken identity” is a huge barrier to correct research. To cite an example from literature, we can see the HeLa cells that were isolated from Henrietta Lacks’s (a 30-year-old African-American woman) cervical cancer biopsy. Several cell lines have been reported to be contaminated with HeLa cells. An article published in The Archives of Pathology & Laboratory Medicine reported that many mouse cell lines and breast cancer lines turned out to be HeLa cells!

Another example of reportedly misidentified cells was reported by Phuchareon and team in 2009 in Plos One. Cell lines of adenoid cystic carcinoma (ACC) that target the salivary glands have been established and are used routinely to understand the biology of this malignancy with a 10-year survival rate of 40%. Short tandem repeat (STR)-based genetic analysis of 6 such cell lines revealed something else! ACCNS and CAC2 lines were contaminated with murine cells, ACC2, ACC3, and ACCM were found to be contaminated with HeLa cells and urinary bladder cells were seen in the ACCS cell line!

Research by Di Girolamo and team in 2016 in Investigative Ophthalmology & Visual Science revealed that an SV40-Transformed Human Corneal Epithelial Cell Line contaminated primary human corneal epithelial cells. The team originally wanted to assess how viable human cornea cells were in storage banks. One such approach of maintaining corneas was submerging the corneas in Eye Bank Organ Culture Media (EBOCM) at 30°C to 37°C termed as organ culture preservation. While most samples displayed viability, 2 samples showed smaller cells with high proliferation.

Phenotypic analysis revealed that these cells were K3 positive (corneal marker) and also contained K15 (stem cell marker) with no antigens that stimulate the immune response. However, as these cells showed non-corneal primary cell-like expansion, they were tested for viruses. As previous work in the lab involved the use of the SV40-immortalized HCEC line, tests for SV40 revealed the presence of this virus in the primary corneas! This was in turn attributed to poor culture technique or accidental inoculation in wrong bottles.

As much energy and resources are wasted due to such contamination, the researchers insist on the importance of authenticating Cancer Cell Line routinely. Additional points to maintain good primary culture without contamination include:

  1. Unused materials are to be discarded
  2. At one time, only one type of cell culture is warranted. If different cells are to be used, separate culture laminar hoods and incubators are to be used.
  3. Avoid the use of reagents used by colleagues
  4. Authentication of cells at routine intervals is important.

Thus, while primary culture are vital tools to study biology, pathogenesis, and screening, avoiding contamination too is vital so that research can continue without a break to unearth the deeper secrets of biology.


Lucey BP, Nelson-Rees WA, Hutchins GM. Henrietta Lacks HeLa cells, and cell culture contamination. The Archives of Pathology & Laboratory Medicine. 2009; 133: 1463–1467.

Phuchareon J, Ohta Y, Woo JM, Eisele DW, Tetsu O. Genetic profiling reveals cross-contamination and misidentification of 6 adenoid cystic carcinoma cell lines: ACC2 ACC3, ACCM, ACCNS, ACCS, and CAC2. PLoS One. 2009; 4: e6040.

Nick Di Girolamo, Sharron Chow, Alex Richardson, Denis Wakefield; Contamination of Primary Human Corneal Epithelial Cells With an SV40-Transformed Human Corneal Epithelial Cell Line: A Lesson for Cell Biologists in Good Laboratory Practice. Investigative Ophthalmology & Visual Science. 2016;57(2):611-616.

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