Treatment of mycoplasma contamination in cell cultures with Plasmocin

Abstract

A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

Figure 1

Treatment protocol for Plasmocin. The reagent is added to the medium at a final concentration of 25 μg/mL on the days indicated by arrows.

Figure 2

Relative treatment efficiency of antimycoplasma antibiotics. The portions given for Plasmocin include the results from second approaches.

Figure 3

Agarose gel showing the results of a mycoplasma detection PCR. Two PCR reactions were performed per sample: one with the sample only and one with the sample and an internal control DNA added at a limiting dilution. The wild-type PCR product is represented by the lower band with a size of approximately 520 bp. The upper band is the amplification product of the internal control DNA (986 bp). (1) 100 bp ladder, (2) mycoplasma-negative cell line, (3) mycoplasma-negative cell line with internal control, (4) A. laidlawii-contaminated cell line, (5) A. laidlawii-contaminated cell line with internal control, (6) M. hyorhinis-contaminated cell line, (7) M. hyorhinis-contaminated cell line with internal control (the intermediate band at approximately 750 bp is a hybrid of a 520 bp and a 986 bp product), (8) internal control only, (9) positive control only, (10) positive control with internal control, (11) water control, and (12) 100 bp ladder.

Figure 4

Approach to treatment of mycoplasma-positive cell cultures with specific antibiotics. The scheme was adapted from [13].