Mycoplasma Contamination in Cell Culture

When was the last time you tested? Not yourself, but your cell culture? Mycoplasma contamination is a well-known but still often ignored problem in cell culture. Currently, only about 30% of cell culture labs test for mycoplasma. Read about the damage mycoplasma can cause and how we can help you with Lonza's MycoAlert^TM products.

WHAT EFFECTS CAN MYCOPLASMA CONTAMINATION HAVE ON CELLS?

The effects on eukaryotic cells can be manifold. Here are a few examples:

• Inhibition of cell metabolism
• Induction of chromosomal and morphological changes
• Changes in DNA, RNA and protein synthesis
• Increased sensitivity to inducers of apoptosis
• Changes in membrane antigenicity
• Changes in transfection efficiency
• Influence on virus production
• Increased cell death rate

HOW CAN YOU DETECT MYCOPLASMA CONTAMINATION?

There are a wide variety of options, most of which are PCR-based. Lonza's MycoAlert is a simple biochemical test that takes 20 minutes and uses the activity of enzymes in mycoplasmas for detection. The presence of these enzymes allows easy and reliable detection of mycoplasma in the sample being tested. Live mycoplasmas are lysed, and the enzyme reacts with the MycoAlert substrate, resulting in a conversion of ADP to ATP. The level of ATP in the sample is measured by a luciferase reaction. There are two reading steps, one before and one after the addition of the MycoAlert substrate. From these two readings, a ratio can be calculated that provides information about the potential presence of mycoplasma. If the sample is clean, there is no increase in ATP level. If mycoplasmas are present in the sample, there is a clear increase in the ATP level resulting in an enhanced light response. Lonza also offers Lucetta2, a suitable luminometer.

CAN MYCOPLASMAS ALSO GROW IN THE ABSENCE OF CELLS?

There are indications of this; as a rule, mycoplasmas grow in the presence of cells. Lonza's MycoAlert PLUS Kit can also detect contamination in cell-free media, sera and additives.

CAN MYCOALERT DETECT THE ENTIRE SPECTRUM OF MYCOPLASMAS?

The mycoplasma-specific metabolism detected by MycoAlert is present in all species of the Mollicutes (Mycoplasma, Acholeplasma, Entomoplasma and Spiroplasma), with the exception of Ureaplasma, which usually plays no role in cell culture. Of all infections, 95% are caused by M.orale, M.arginii, M.fermentans, M.salivarum, M.hyorhinis and A.laidlawii. MycoAlert can detect all these species, plus at least 44 others that have been tested for to date.

Do bacteria lead to a false positive result?

No. The reagent included in the kit for lysis is not strong enough to attack bacteria with cell walls or yeasts. Also, the enzymes used are not normally present in bacteria or yeast but are present in Mollicutes. Bacterial contamination is usually noticeable due to a rapid increase in turbidity of the culture medium. Mycoplasma infection does not cause turbidity and therefore the need for a test method such as MycoAlert.

ARE POSITIVE CONTROLS AVAILABLE FOR MYCOALERT?

Yes, an  Assay Control Set is available. This contains a lyophilized positive control (1mL) and 2mL of assay buffer to reconstitute. The buffer also serves as a negative control. The controls have been specifically designed for the MycoAlert kit. The formulation of the positive control is protected but does not contain mycoplasma. The two controls are treated as one sample in the assay, the positive control gives a ratio of >1 for the two measurements, the negative control <1.

WHAT SAMPLE VOLUME FROM CULTURE SUPERNATANT IS NEEDED FOR ACCURATE MEASUREMENT RESULTS?

According to standard protocol, 2mL of supernatant should be taken and centrifuged. A further 100µL should be used.

WHICH SAMPLE SHOULD BE USED? CAN IT BE STORED?

The MycoAlert assay requires cell-free culture supernatant. The supernatant must be centrifuged at 200xg or 1500rpm for 5 minutes to remove all cells. Cells would increase the background signal, decrease sensitivity, or confound results. Optimally, the supernatant should be processed as quickly as possible. If storage is necessary, it must be at 4°C if the sample is tested within one day. A supernatant stored at -80°C can be used for up to 6 months. In this case, the supernatant should be frozen immediately after centrifugation and thawed without additional heat if needed. The supernatant should then be left at room temperature for at least 15 minutes before testing.

CAN it WORK IN a 96-WELL FORMAT?

Yes. The standard protocol and the volume required are no different from the single-tube version. Ideally, the kit for 100 tests should be used.

WHICH MICROTITER PLATES ARE RECOMMENDED?

For example, Lonza recommends the Corning-Costar Plate with the article number 3912, which guarantees very low background signal and optimal results in a plate luminometer.

IS IT POSSIBLE TO QUANTIFY THE RESULTS WITH THE MYCOALERT?

No, this is not possible. The assay is designed for a yes/no statement.

HOW IS THE ASSAY VALIDATED?

The MycoAlert assay is for ‘Research Use Only’.

WHAT IS THE DIFFERENCE BETWEEN MYCOALERT AND MYCOALERT PLUS?

Both assays are based on the same principle. The MycoAlert PLUS Kit generates a higher light output and can therefore be used on a larger number of readers.

WHAT CAN BE DONE WITH CELL CULTURES IN CASE OF MYCOPLASMA CONTAMINATION?

If contamination occurs and the sample absolutely cannot be discarded,  MycoZap^TM  – the Mycoplasma Elimination Reagent – provides a way to eliminate the contamination with minimal toxic effect on the cells. MycoZap eliminates mycoplasma through a combination of antibiotic and antimetabolic agents. This combination works with a high degree of reliability. MycoZap is effective against Mollicutes, including all species of Mycoplasma, Acholeplasma, Spiroplasma and Enteroplasma in cell cultures. In general, cell cultures should be tested with the MycoAlert Mycoplasma Detection Kit at regular intervals of 4-6 weeks after elimination.

Source: Lonza Resource Notes

June 15, 2021