September 1, 2007 (Vol. 27, No. 15)

Assay Enables the Testing of Cultures to Detect Infections at an Early Stage

Winthin the class of Mollicutes, there are Mycoplasmas, Ureaplasmas, and Acholeplasmas. This group represents the smallest and simplest form of prokaryotes. They lack a rigid cell wall and are therefore not susceptible to common antibiotics such as penicillin and its analogues. They have developed by degenerate evolution from gram-positive bacteria resulting in the loss of a substantial part of their genetic information. It is this limited coding capacity that has dictated the need for a parasitic way of life.

More than 120 Mollicute 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 six species that account for 95% of all Mycoplasma infections: M. orale, M. arginini, M. fermentans, M. salivarum, M. hyorhinis, and A. laidlawii.

It has been reported that 15–35% of all continuous cell lines are infected with Mycoplasma, a major cause of infection due to cross contamination from other cell lines introduced into laboratories.

Mycoplasmal infections rarely produce turbid growth or obvious cell damage and so cannot be seen under the microscope. Viable Mycoplasma can be recovered from work surfaces seven days after inoculation, due to their small size (0.2–0.8µm). They can also pass through bacterial-retaining filters. At their maximum population phase there can be as many as 108, Mycoplasma/mL of supernatant, at a ratio of 5:1 with the host cells.

Mycoplasma are capable of altering almost every property of an in vitro culture. As the Mycoplasma compete with the cell lines for the nutrients in culture media, one of the first signs is a slow-down in cell proliferation and changes in cellular responses and gene expression.

The transfection efficiency of the cells may be compromised, and the cells can become more sensitive to inducers of apoptosis. Mycoplasma also possess nucleases that can cleave the host DNA to give the appearance of apoptosis. A Mycoplasma infection can induce gene expression.

The most common induced changes include disruption of nucleic acid synthesis leading to chromosomal breakage, multiple translocation events, and even a reduction in chromosome number.

Their metabolism can also cause aberrant results with tetrazolium assays, masking any cytotoxic effects of compounds and causing shifts in EC50 values. This is the result of the ability of certain Mycoplasma species to reduce the tetrazolium salts and increasing colored product.

Their resistance to antibiotics and ability to pass through filters means that they can evade the typical precautions of cell culture technique. As a result of the negative impact of having these contaminations going undetected, it has become evident that continuous screening is essential for any cell culture laboratory.

Figure 1

Contamination Testing

Lonza’s ( MycoAlert® Mycoplasma Detection assay is a generic biochemical test for Mycoplasma and other mollicutes. It offers the possibility to test cultures for mycoplasmal contamination whenever the cells are passaged and gives the user a profile of their cells, allowing them to spot infections at an early stage (Figure 1).

The MycoAlert Assay detects the activity of mycoplasmal enzymes, which generate ATP. When provided with specific substrates, these enzymes are able to convert ADP to ATP, which is then detected using the bioluminescent luciferase/luciferin reaction. The kinetics of this reaction are illustrated in Figure 2.

The MycoAlert Assay is simple to perform. First, the MycoAlert Reagent, containing the bioluminescent detection system and a detergent that will lyse any Mycoplasma present, is added to the test sample. The sample is incubated at room temperature for five minutes, establishing an ATP baseline measurement. Then, a one-second integrated light reading is taken (Reading A in Figure 2).

Next, the MycoAlert Substrate is added. This reagent contains the specific substrates for the mycoplasmal enzymes. After a 10-minute incubation at room temperature, a second reading (Reading B in Figure 2) is taken. By dividing Reading B by Reading A, a ratio is obtained.

If Mycoplasma are present, the enzymes convert ADP to ATP, shown by a dramatic increase in the number of RLUs (red line). Hence, the value of the ratio will be above one, indicating a positive sample. If no Mycoplasma are present, the baseline ATP level will gradually drop, as ATP is consumed by the luciferase/luciferin reaction (blue line). In this case, the value of the ratio will be below one, indicating a negative sample.

The MycoAlert Assay has been optimized to work with cell culture samples under normal culture conditions. It does not require any modifications to be made to the samples and can be conducted in the presence of antibiotics and colored culture medium containing serum. To date, 39 species have been tested and detected successfully by MycoAlert (Table 1). The tool will detect mollicutes of mammalian, avian, insect, and plant origin.

Being a biochemical assay that detects the activity of specific mycoplasmal enzymes, false-positive results will not be generated by eukaryotic cells that do not possess the same enzymes. The use of a selective lysis procedure means that any prokaryotes other than mollicutes that may express the same enzymes will not be detected (Table 2).

MycoAlert was used to monitor the progression of a Mycoplasma infection. K562 (human leukaemia) cells were infected with two common species of Mycoplasma and monitored regularly by removing a small sample of the culture supernatant. The infection is apparent less than three days after infection (Figure 3).

Routine monitoring of all cell lines in culture showed a covert Mycoplasma infection that was subsequently identified as M. hyorhinis. The cell lines were definitely identified as being positive or negative for Mycoplasma by the use of the MycoAlert Assay (Figure 4). The results were verified by PCR.

MycoAlert Mycoplasma detection assay offers the opportunity for routine testing of cell cultures and has been designed to easily incorporate into standard tissue culture regimes. The assay can be completed in approximately 20 minutes and provides a definitive answer. It requires no specialist treatment of the cells prior to testing and uses spent media, which would be discarded with no need to include precious cells.

Figure 2

Figure 3

Figure 4

Claire Scholfield, Ph.D., is research
scientist, Anne Cox, Ph.D., is research scientist, and Anthony Pitt, Ph.D.,
is program director, research products, at Lonza Nottingham.
Phone: 44 115 912 4346
E-mail: [email protected]

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