15 Points For Primary Cultures In Biomedical Research

In order to minimize problems of misidentification that can lead to research being retracted and the loss associated due to contamination. The following pointers can be used as a reminder to facilitate good practices for primary cultures in biomedical research:

1. The derivation of culture from fresh human tissue involves the first step of obtaining ethical review approval or donor’s consent (as applicable to the experiment). This should include the details such as the origin of the sample, pathology, histopathology report, identification numbers and clinical history apart from the basic details of the patient/donor.
2. A piece of the sample used to establish primary cultures should be processed as soon as possible. Another aliquot should be frozen that can serve as proof for the authenticity of the culture. The authenticity can be checked using sequencing or karyotype studies and short tandem repeat (STR) profiling.
3. Simultaneously, histopathological analysis of the sample is warranted to ensure that the sample contains the pathology or disease or genetic condition being studied.
4. While antibiotics are added to primary culture media, their removal is suggested as soon as possible and routine testing for mycoplasma must be done. Maintenance of records of how the test was performed is suggested.
5. An important point to be followed in the lab is keeping the manipulation of living cells separate from media preparation.
6. While preparing primary cultures in biomedical research, images of the various stages of growth are to be taken and stored. These serve as a reference for future work.
7. In a culture lab, the use of Liquid nitrogen apart from being vital is a source of danger. For example, a 2007 published article in Science, reported the death of a worker in Australia due to liquid nitrogen-induced asphyxiation. This dictates that the relevant safety is followed in terms of placing and handling liquid nitrogen cylinders to avoid cold bites or explosions.
8. As the status of a tissue taken from a donor may not be in sync to that of the donor in terms of microbiology, samples such as secretions, blood, cells and body fluids should be handled in Containment Level 2 in a Class II MSC.
9. Vaccinating (tetanus and Hepatitis B) the lab members who handle tissues and cell lines is recommended.
10. Batch variation in reagents and sera can be minimized by bulk buying.
11. Repeatedly thawing and freezing protein solutions such as trypsin or sera is not recommended, to aliquot them according to use is a good practice.
12. When using 10 × concentrated media from suppliers, care must be taken that there are no alterations to the pH or any precipitate in the concentrate.
13. While working in the microbiological safety cabinet (MSC), apart from maintaining asepsis, fluids should be handled slowly to avoid aerosols and rapid movements are to be avoided. Unless absolutely vital, the use of a burner in an MSC is to be avoided to avoid interference to the air currents.
14. Regular checks and cleaning of incubators and autoclaves that are integral in cell culture are vital.
15. Troubleshooting in the event of an issue can help reach solutions quickly. For example, for altered cell growth in culture, the issue can be traced to the media, culture conditions and the cells. The question ‘what is new?’ in terms of reagents or batches or protocols can greatly assist arriving at a solution.

The use of guidelines with foresight can help avert issues in cell culture labs. This can ensure the smooth functioning of the lab to supply cell cultures that are known to be an integral part of research.

References:

Finkel E (2007) Research safety. Inquest flags little-known danger of high-containment labs. Science 316 (5825): 677.

Geraghty, R., Capes-Davis, A., Davis, J. et al. Guidelines for the use of cell lines in biomedical research. Br J Cancer 111, 1021–1046 (2014).