I need to thaw my freezing cancel cell line,and i do not want to check the contamination of mycoplasma,therefore i want to use a clean up mycoplasma kit.
Do you have any suspections that your cells might be infected? Are these standard cells or a line made by your lab? In my experience mycoplasma is usually hard to get rid off, so if you have it and have the chance, I would get fresh (and mycoplasma free) cells from a repository. Otherwise test the cells with PCR, this is fast and reliable, you have to do it anyway routinely.
Coming to the question: I would treat cells with anti-mycoplasma reagents if not absolutely necessary. Cells don't like this very much. When we took cells from the liquid nitrogen storage, we only put them in culture.
Eric, we have a well-organized way of dealing with Mycoplasma contamination/elimination in our lab; to decide what to do, you need to be sure of the status of the frozen cell lines. If you are not sure of their contamination/sterile status, it is a good practice to check them as you thaw. Treat or discard those that are contaminated and keep the rest for your work.
However, many times, the culture media may be a source of contamination or the handling itself could lead to contamination: check for contamination/sterility just before freezing them back for future use.
In my experience, most cell lines are contaminated with Mycoplasma during culturing or assaying, particularly those that undergo some sort of long term propagation for any use.
Cheers.
If they where free of mycoplasma before freezing, they still should be ok. I know there are reports of infections in the liquid nitrogen tank, but I doubt it. We had the case that some guy froze down infected cells (without knowing) and eventually found out they where. There were no problems for the rest of the cells in the tank (not even from the same box).
So I would put them in culture, work with them and test them at some point. You can do this with primers you order by yourself, I can look up the reference, if you (or anybody else) are interested.
Thanks, Christian Praetorius, you just confirmed something I have always reminded my colleagues: cross contamination in the liquid nitrogen tank is an impossible hypothesis. I know that people forget what they froze if they were actually clean before the freezing.
Please, share the primers reference, and, if possible, I am currently looking for a source of Mycoplasma pirum control and Acholeplasma laidlawii to use in my PCR assays. Have any idea where I can but from?
Thanks.
Sure. We used the primers which had been published in:
Uphoff, C. C. and H. G. Drexler (2004). "Detecting Mycoplasma contamination in cell cultures by polymerase chain reaction." Methods Mol Med 88: 319-326.
We got the positive control mentioned in this paper directly from the author (simply by writing them an email and asking for it and later transformed them into bacteria). For this we adopted the protocol provided for our lab (I attached it to this post). As a positive control we used the supernatand of a mycoplasma positive cell line (heat inactivated).
As the leader of the PDA mycoplasma Task Force I could recommend to you the PDA Technical Report No. 50 Alternate methods for mycoplasma testing, 2010. Also the paper published i n 2004 describes in detail the PCR method that has been accepted by the FDA, EMA, Japan regulatory agency and the Canadian regulatory agency. The paper gives universal primers that detect over 15 different species.. Eldering,J.A., Felton, C., Veilleux, C.A., Potts, B.J., Biologicals 2010, 38,183-193.. There was also special section in the March 2010 issues of Biologicals on mycoplasma that includes the validation reports from three companies who used this assay or a variation of it for successful regulatory acceptance. Biologicals vol 38, No 2, March 2010 pp 181-248. If you intend on using your cells for clinical trials you should be sure to follow the guidelines in Points to consider in the Characterization of cell lines used to produce biologicals. US FDA Silver Spring MD March 1993.
The best source of A. laidlawii is from the ATCC. To prevent a mycoplasma contamination from reagents make sure to use serum, media that claim to have been tested for mycoplasma. Serum will often have mycoplasma but can be inactivated even at 40C. Always use a class II hood to do your work and have operators wear disposable gowns and sterile gloves when working with the cells. It is best to keep mycoplasma out rather then to treat the cells later,. I agree that the mycoplasma removing agents can be harsh on cells and in my experience I needed to keep the reagent in my media for three months to assure that the mycoplasma is really removed.. Good luck
Barb Potts
Potts and Nelson Consulting, LLC.
@Barbara Potts: Despite your claims, I never had trouble with mycoplasma in serum.
You are in luck. If you heat inactivate the serum at 50C for 45 minutes you will inactivate mycoplasma. Serum (bovine and fetal) are the main cause of mycoplasma contamination in the biotech industry.
Barb Potts
@Barbara: I used sera from a lot of sources, all had been certified mycoplasma free. We tested them regulary along with our cultures and never found problems. I wouldn't bluntly heat sera without reason (complement deactivation would be one), because this might affect the quality of the serum and the activity of the factors in it.
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