- Mycoplasma contamination is a prevalent issue in cell culture laboratories, posing significant challenges. However, a comprehensive understanding of mycoplasma contamination sources, coupled with good aseptic cell culture techniques and regular mycoplasma testing of cell lines, can substantially reduce the risk of contamination.
- The primary sources of mycoplasma contamination in cell culture are contaminated cell lines, laboratory personnel, and culture medium. Additionally, the cell culture room, liquid nitrogen storage, laboratory instruments (e.g., laminar flow hood, water bath, incubator), and unsterile labware can also serve as potential sources of mycoplasma contamination.
- Currently, contaminated cell lines are regarded as a major source of mycoplasma spread to other cell lines within cell culture laboratories. Therefore, all primary cultures and newly acquired cell lines should be considered potentially contaminated until tested and confirmed to be mycoplasma-free using standard detection methods. Furthermore, an ongoing cell culture can become contaminated with mycoplasma through various means. Contaminated cultures can not be identified through regular light microscopy, and consequently, can serve as a significant source of mycoplasma spread, particularly when a shared cell culture medium is used to maintain both contaminated and mycoplasma-free cell lines.
- Laboratory personnel, especially less experienced junior researchers, are also considered a major source of mycoplasma contamination. Factors such as poor personal hygiene, bad aseptic techniques, and activities like discussion (generate aerosols) within cell culture labs can all introduce mycoplasma contamination to cell lines. Human-borne mycoplasma species, such as M. orale which naturally colonize the human oral cavity and oropharynx, have been detected in several cell lines.
- Another crucial source of mycoplasma contamination is the cell culture medium. It is important to note that filtration through a 0.2 μm filter unit cannot remove mycoplasma; therefore, it is critical to prepare the culture medium using high-quality constituents, including high-quality water, to minimize the risk of mycoplasma introduction. In the past, serum was a primary source of mycoplasma contamination, but many reputable suppliers now provide mycoplasma-tested serum. Furthermore, the prepared culture medium can also be contaminated. The risk of contamination is particularly high if the same culture medium is shared among multiple researchers or used for culturing many cell lines. This practice can facilitate the spread of mycoplasma from a contaminated cell line to other cultures, leading to widespread contamination.
- Equipment used in cell culture labs can also be a source of mycoplasma contamination. Spillage of media from a contaminated culture can cause the spread of mycoplasma to healthy cell lines. Additionally, poor maintenance of the cell culture laboratory can also invite mycoplasma contamination. Cell culture labs must be carefully maintained in a clean state, free from any insects, animals, or dust.
- The excessive use of antibiotics in cell culture can further indirectly enhance the incidence of mycoplasma contamination. The overreliance on antibiotics can promote poor aseptic techniques among cell culture lab users which will increase the risk of mycoplasma introduction to cell lines.
- Mycoplasma can also survive in liquid nitrogen without the need for a cryoprotectant, making it a potential means of spreading contamination. Therefore, to mitigate this risk, it is advisable to store frozen cell culture vials in the vapor phase of liquid nitrogen, rather than the liquid phase.
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