One of the most common contaminants present in cell culture laboratories are mycoplasma. A conservative estimate states that between 15-35% of all continuous cell cultures are contaminated with mycoplasma1, some estimates are even higher (up to 80 % in some countries2.
Mycoplasmas belong to the family Mollicutes, which includes Acholeplasma, Ureaplasma and other species. However the term Mycoplasma is most often used as a cover-all'. More than 180 species have been identified of which 20 distinct Mycoplasma and Acholeplasma species from human, bovine and swine have been isolated from cell culture. There are 6 species that account for 95% of all mycoplasma infections (M.orale, M.arginii, M.fermentans, M.salivarum, M.hyorhinis and A.laidlawii). Mycoplasmas are widespread in nature as parasites of humans, mammals, reptiles, insects and plants. They are the smallest and simplest self-replicating prokaryotes, they lack a rigid cell wall and are surrounded by a single plasma membrane. They are dependent on their hosts for many nutrients as their biosynthetic capabilities are limited.
Typical routes of infection are cross contamination from untested infected cells (e.g. via aerosols generated during pipetting, use of same media bottles, handling of more than one cell type at one time), contaminated materials, contaminated donor tissue (<1%) or direct infection from the researcher. The primary source is normally cross contamination from infected cultures.
Effects of mycoplasma contaminations on cell cultures
Mycoplasma grow slowly and do not kill the cells outright but affect various cellular parameters1,3,4,5,6,7,8,9,10,11, e.g.
- Increased sensitivity to apoptosis
- Chromosomal aberrations
- Change of gene expression patterns
- Changes in cell membrane antigenicity
- Inhibition of cell growth
- DNA fragmentation due to mycoplasma nucleases
- Compromised production of viruses
- Inhibition of cell metabolism
- Reduction of transfection efficiencies
- Cell death
Certain mycoplasma species are able to reduce tetrazolium salts causing aberrant results with tetrazolium assays, and so could mask any cytotoxic effects of compounds and cause shifts in IC50 values.
Thus, mycoplasma contaminations can seriously impact the reliability, reproducibility, and consistency of experimental results, representing a major problem for basic research as well as for the manufacturing of bioproducts. Standard testing for mycoplasma is an important quality control.
Detection of mycoplasma
Even at very high concentrations (> 107 cfu/ml) mycoplasma are not visible by microscopy. They do not cause visible changes in the growth media that are commonly associated with bacterial and fungal contamination, such a turbidity or pH changes1. Therefore contaminations are very difficult to detect and the presence of mycoplasma can remain undiscovered for months.
As mycoplasma compete with cells for the nutrients in culture media, one of the first visible signs is a slow down in cell proliferation. Other indications of contamination include cell aggregation, morphological changes or poor transfection efficiencies with cells that originally transfected well.
References
1) Drexler HG, Uphof CC (2002). Cytotechnology 39: 75–90
2) Koshimizu K, Kotani H (1981). In: Procedures for the Isolation and Identification of Human, Animal and Plant Mycoplasmas (Nakamura, M., ed.), Saikon, Tokyo, 87-102.
3) Gong H, Zölzer F, von Recklinghausen G, Rössler J, Breit S, Havers W, Fotsis T, Schweigerer L (1999). Biochem Biophys Res Comm 261: 10-14.
4) Ben-Menachem G, Mousa A, Brenner T, Pinto F, Zähringer U, Rottem S (2001). FEMS Microbiol Letters 201: 157-162
5) McGarrity MF, Vanaman V, Sarama J (1984). In Vitro 20: 1-18
6) Sokolova IA, Vaughan ATM, Khodarev NN (1998). Immunol Cell Biol 76: 526-534
7) Doersen CJ, Stanbridge EJ (1981). Mol Cell Biol 1: 321-329
8) Stanbridge EJ (1971). Bacteriological Reviews 35): 206-227
9) Darin N, Kadhom N, Brière JJ, Chretien D, Bébéar CM, Rötig A, Munnich A, Rustin P (2003). BMC Biochem 4:15
10) Rottem S (2003). Physiol Rev 83: 417-432
11) Miller CJ, Kassem HS, Pepper SD, Hey Y, Ward TH, Margison GP. (2003). Biotechniques 35:812-814
