Mycoplasma Elimination
The contamination of cells with mycoplasmas is a very common problem, even though it often remains unnoticed since no cloudiness appears in the cell culture. Nevertheless, the contamination often causes significant biochemical changes as well as changes in the immunological properties of the cells. For both biological and economical reasons, it is important to eliminate mycoplasmas from cell cultures used for basic research, diagnosis, quality testing, and biotechnological production. Since mycoplasma-infected cells cannot always be discarded, many complicated methods have been suggested for the elimination of the mycoplasmas.
PromoKine is now offering two highly effective methods to get rid of mycoplasma contamination:
- Mycoplasma-EX, a combination of two solutions (non-antibiotic/antibiotic) with extremely low cytotoxicity. While the antibiotic efficiently inhibiting mycoplasma growth, the non-antibiotic solution directly kills mycoplasmas fast and very efficiently. It can be used to clean most eukaryotic cell cultures or virus stocks directly. Cultures are free of mycoplasmas after only four passages!
- BIOMYC, a combination of three different antibiotics which have been shown to be very effective in the elimination of mycoplasma species that account for 90% of the contamination found in cell cultures. When used according to our instructions, no cytotoxic effects will occur. The BIOMYC solutions are a very cost-effective way to get cell cultures free of mycoplasmas - however, it is more time-consuming and labor-intensive compared to the Mycoplasma-EX treatment.
Mycoplasma-EX
The most effective reagent against mycoplasmas
Mycoplasmas in cell culture are - among other things - affecting cell growth and cell physiology seriously leading to unsatisfying and less significant results. The increasingly widespread use of more sophisticated and sensitive methods for the detection of mycoplasma contamination in cell cultures has resulted in contamination being detected in numerous cultures. This raises the issue of how to eliminate mycoplasma contamination. Naturally, the ideal solution is to discard the contaminated cells. However, if the valuable cells that are stored in liquid nitrogen are also contaminated, a solution is required for eliminating the mycoplasmas and preparing a new cell bank, particularly if the cells are unique and the result of extensive work. The most commonly used method for elimination, inactivation, or suppression of mycoplasmas in cell cultures is treatment with distinct antibiotics. In general, antibiotic therapies do not result in long-lasting, successful elimination. Also, the cytotoxic properties of antibiotics can cause undesirable side effects on eukaryotic cells and may facilitate the development of resistant mycoplasma strains.
The new Mycoplasma-EX method for mycoplasma elimination overcomes all of these drawbacks combining a non-antibiotic and a thoroughly adjusted antibiotic treatment resulting in unique features as compared to other methods and products. Mycoplasma-EX is the first biological reagent that eliminates mycoplasmas by directly killing them, and not just by inhibiting their growth. It is the only anti-mycoplasma agent that can be used to clean virus stocks and most eukaryotic cell cultures directly while showing an extremely low cytotoxicity. Mycoplasma-EX has been shown to be effective with only one treatment, destroying mycoplasmas permanently within 2-3 hours using the non-antibiotic Initial Treatment Reagent only. When using in addition the antibiotic Succession Treatment Reagent, the teatment time extends to - depending on the cell type - two to four weeks. Mycoplasma-Ex is applicable for most cell lines (e.g. Vero, BHK21, GBK, ML, Hep2, 293, CRFK, H9, Molt4, MT-4, Jurkat) as well as virus stocks (e.g. SHV-1, BHV-1, HSV-1, VSV, SFV, FCV, MEV). Success of mycoplasma elimination has to be determined after the initial and - if required - succession treatment using a sensitive mycoplasma detection kit (e.g. our PCR Mycoplasma Test Kits).
Benefits of Mycoplasma-EX:
- highly effective against M. orale, M. hyorhinis, M. hominis, Acholeplasma laidlawii, M. arginini, M. fermentans, and other mycoplasma species usually encountered as contaminants in cell cultures
- combined non-antibiotic and antibiotic treatment
- significantly reduced treatment time
- ease of use
- temperature stable, ready-to-use solution
- no additional dosing during the treatment necessary
- low cytotoxicity: kills mycoplasmas very efficiently - but is safe for cells
Mycoplasma-EX is a sterile, ready-to-use solution, aliquoted per tube for single use. Store the original container at temperatures of +2 to + 8°C.
Using Antibiotics to Disinfect Cell Cultures
A number of effective methods for the elimination of mycoplasma contamination in cell cultures have been published, such as:
- treatment with specific hyperimmune serum (antibodies).
- passage of contaminated cells in thymus-deficient mice.
- exposure to analogs of nucleic acids that prevent reproduction of mycoplasma.
- treatment with antibiotics.
- exposure of contaminated cells to mouse macrophages.
- a technique that combines growing cells on soft agar and treatment with antibiotics.
The preferred method in terms of simplicity is treatment with antibiotics, which do not damage or alter cells.
Antibiotics such as penicillin, which attacks bacterial cell walls, are ineffective in this instance, since mycoplasmas lacks a true cell wall. Several antibiotics eliminate mycoplasmas effectively, such as: Tylosin, Neomycin, Tetracycline and Gentamycin. However, the efficacy of these antibiotics is restricted to specific mycoplasma species and frequently only reduce the concentration of mycoplasmas rather than disinfect the cell culture. Hence, as soon as treatment is concluded, contamination will recur.
Two methods are recommended for treating contaminated cells with antibiotics. The first is based on alternating treatment with two types of antibiotics, and the second on treatment with one type of antibiotic.
BIOMYC Antibiotic Solutions
BIOMYC-1 & BIOMYC-2
BIOMYC-1 is a solution based on the antibiotic tiamutin which is produced by the fungus Pleurotus mutilus. BIOMYC-2 is a solution based on the antibiotic minocycline, which is a tetracycline derivative.
These two antibiotics have been shown to be very effective in eliminating the mycoplasma species frequently present in contaminated cell cultures and are generally used sequentially in combination. Following our protocol, the mycoplasmas do not develop resistance to these antibiotics - a common occurrence with other antibiotic treatment methods. The treatment is based on alternating use of the two antibiotics: four days with tiamutin followed by three days with minocycline, and repeating the cycle 2-3 times. On completion of the treatment, the cells should be grown on media without antibiotics for at least two weeks and then re-tested for the presence of mycoplasmas. If the culture test is positive, the course of treatment should be repeated.
Instructions for Use
- Do not use the two solutions together, but rather sequentially.
- Add 1 ml BIOMYC-1 to 100 ml medium, and maintain the contaminated cells in this mixture for 4 days. Any fresh medium added should also contain BIOMYC-1.
- After 4 days, add 1 ml BIOMYC-2 to 100 ml fresh medium, and maintain the cells in this second mixture for 3 days.
- The above, together, are considered as one treatment cycle. It may be necessary to repeat this cycle 2-3 times.
- During the process, the cells can be tested for mycoplasma contamination, and results can then be used to shorten the process when possible.
BIOMYC-3
BIOMYC-3 is based on the antibiotic ciprofloxacin, which is a member of the fluoroquinolone group and inhibits synthesis of the enzyme DNA gyrase which is responsible for supercoiling in bacterial DNA. Many mycoplasma species have been found to be sensitive to BIOMYC-3, including A. laidlawii, M. orale, M. hyorhinis, M. fermentans, and M. arginini. These species are responsible for most mycoplasma contamination in cell culture. At the concentrations recommended for use, no cytotoxic effects have been found so far, and the treatment is quite easy to perform.
The treatment consists of adding the antibiotic to the growth medium for 12 days, and changing the medium every three days. On completion of the treatment, the cells should be grown on media without antibiotics for at least two weeks and then re-tested for the presence of mycoplasma. If the culture tests positive, the course of treatment should be repeated. Treatment with ciprofloxacin does not damage or alter the cells. This antibiotic should not be used repeatedly in cell cultures; otherwise, the mycoplasmas are liable to develop resistance to it.
Instructions for Use
- Add 1 ml BIOMYC-3 to 100 ml medium.
- Continue the treatment for a total of 14 days, while changing the medium (containing BIOMYC-3) every 2-3 days.
- Retain the cells in the growth medium for an additional 14 days before re-testing for mycoplasmas.
Reference
Schmitt, K. et al., J. Immunol. Methods, 109:17-25 (1988).
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See also: PromoCell's cell culture products - particularly cells and media for normal human cell culture. >More Information
Note: Mycoplasma safety is also topic of a training course of our PromoCell Academy! >More Information