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Indicator cell lines for the detection of hidden mycoplasma contamination, using an adenosine phosphorylase screening
G T Spierenburg1, A A Polak-Vogelzang, B J Bast
1Department of Clinical Immunology, University Hospital, Utrecht, The Netherlands.
Abstract:
Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in all common mycoplasma species but is considered to have a low activity in mammalian cells. However, using an adoP screening test, we have observed that some cell cultures do possess an intrinsic adoP activity leading to false positive results. Moreover, as a false negative result, we encountered a variant of Mycoplasma orale (identified after cultivation on agar and immunostaining) which was not detectable with the adoP screening in cell culture supernatants and only at low levels in cell lysates. To increase the low signal/noise adoP ratio found there, we used an indicator cell line with low intrinsic activity. Indicator cells were inoculated with the test supernatant and the adoP activity of these infected cells were measured after lysis. The procedure diminished the effect of biological variation in intrinsic enzyme activity between the several cell lines tested. Furthermore, in another mycoplasma infected cell line (with M. fermentans), this infection was only reliably detected using these indicator cells. With this procedure we obtained rapid results which were concordant with those obtained using the time consuming cultivation on agar.
Insights
Mycoplasma contamination in cell cultures can cause false positive or negative results using adenosine phosphorylase (adoP) screening. An improved indicator cell line method enhances detection accuracy and speed for mycoplasma contamination.
Area of Science:
- Cell Biology
- Microbiology
- Biochemistry
Background:
- Mycoplasmas are common contaminants in cell cultures, complicating research, particularly during HAT selection.
- Adenosine phosphorylase (adoP) is a target enzyme for mycoplasma detection, but intrinsic activity in mammalian cells and mycoplasma variants can lead to inaccurate results.
- Existing adoP screening methods can yield false positives due to endogenous enzyme activity and false negatives with specific mycoplasma strains like Mycoplasma orale.
Purpose of the Study:
- To develop a more reliable and sensitive method for detecting mycoplasma contamination in cell cultures.
- To overcome limitations of existing adenosine phosphorylase (adoP) screening tests, including false positives and negatives.
- To establish a rapid and accurate diagnostic procedure for mycoplasma detection in cell cultures.
Main Methods:
- Utilized an indicator cell line with low intrinsic adenosine phosphorylase (adoP) activity to enhance signal-to-noise ratio.
- Inoculated indicator cells with test supernatants from potentially contaminated cultures and measured adoP activity after cell lysis.
- Compared results with traditional agar cultivation methods and tested detection of Mycoplasma fermentans infections.
Main Results:
- The indicator cell line method significantly reduced the impact of biological variation in intrinsic adoP activity across different cell lines.
- A variant of Mycoplasma orale, previously undetectable or detected at low levels, was reliably identified using the indicator cell system.
- Mycoplasma fermentans infection was accurately detected using the indicator cells, a feat not consistently achieved with standard methods.
- The developed procedure yielded rapid results that were concordant with time-consuming agar cultivation.
Conclusions:
- The indicator cell line approach provides a robust and sensitive method for detecting mycoplasma contamination in cell cultures.
- This improved adoP-based screening method offers a faster and more reliable alternative to traditional detection techniques.
- The strategy effectively addresses the challenges posed by intrinsic enzyme activity and mycoplasma strain variability in contamination testing.