Leukemia Research Vol. 16, No. 8, pp. 815~22, 1992. 0145--212(~/92 $.5.00 + 0.(30 Printed in Great Britain. Pergamon Press Lid TREATMENT OF MYCOPLASMA-CONTAMINATED CONTINUOUS CELL LINES WITH MYCOPLASMA REMOVAL AGENT (MRA) SUZANNE M. GIGNAC, CORD C. UPHOFF, RODERICKA. F. MACLEOD, KLAUS STEUBE, MAREN VOGES and HANS G. DREXLER German Collection of Micro-organisms & Cell Cultures, Human and Animal Cell Cultures Collection, Braunschweig, F.R.G. (Received 11 September 1991. Accepted 12 December 1991) Abstract--Thirty-nine continuous adherent or suspension cell lines were treated with a quinolone antibiotic, Mycoplasma Removal Agent (MRA), for the elimination of chronic mycoplasma con- tamination. In preliminary experiments MRA did not show any cytostatic or cytotoxic effects on mycoplasma-free cell cultures in concentrations up to ten-fold the concentration used for mycoplasma eradication. Twenty-eight cell lines (72%) were effectively cleansed of the mycoplasma contaminants by MRA treatment. The persistent removal of the mycoplasma infection was monitored by three mycoplasma detection assays. In seven cell lines (18%) the mycoplasmas were resistant to treatment with MRA. The resistant species was mainly M. arginini followed by M. orale and A. laidlawii; however, other cell lines harboring these species were cured. Four cell lines (10%) which prior to treatment presented with decreased viability and poor or no cell growth were lost during or shortly after the exposure to the antibiotic. If an antibiotic elimination is attempted it is imperative to closely examine the effectiveness of treatment and possible eukaryotic cytotoxicity. The treated mycoplasma- free cells may also no longer express the original features as a result of treatment or the absence of mycoplasma. Key words: Mycoplasma, cell lines, elimination, antibiotics. INTRODUCTION THE in vitro culture of cells has opened many doors in research, and continuous cell lines have become indispensable and powerful tools in biomedical research. However, the use of cell lines has also brought new problems, one of the most serious being contamination with micro-organisms. While infec- tions with bacteria, yeast or fungi will be easily and quickly recognized, contamination with myco- plasmas might escape the attention of the cell cul- turist for a long time unless specific detection assays are employed [1]. Chronic contamination with myco- plasmas can cause a wide variety of effects on their hosts, the eukaryotic cells: for instance interference with cellular parameters measured in experiments; changes of the host cell metabolism; alterations of cell karyotype; or retardation of cell growth. Ulti- mately, the culture might be lost as the cells might cease to proliferate due to a massive, overwhelming infection. Correspondence to: Dr Hans G. Drexler, M.D., DSM- Deutsche Sammlung yon Mikroorganismen und Zell- kulturen, Maseheroder Weg 1B, D-3300 Braunschweig, F.R.G. 815 Mycoplasmas are the smallest self-replicating pro- karyotes. Unlike other prokaryotes they do not have cell walls and are therefore placed in a separate class: mollicutes. Thus, mycoplasmas are neither bacteria nor viruses but share some of the features of both. That mycoplasma contamination of cell cultures is not just an infrequent and innocent nuisance to the researcher is illustrated by the fact that a large per- centage of continuous cell lines in current use is chronically infected: estimates range from 12 to 35% [2, 3]. With regard to our experience we reported that 44 out of 95 (45%) human leukemia-lymphoma cell lines received from outside laboratories were mycoplasma-positive [1]. The most prevalent myco- plasma species are M. arginini, M. fermentans, M. hyorhinis, M. orale and the related Acholeplasma laidlawii [4]. The main focus of mycoplasma control should be on preventing cell culture contamination. It is gen- erally recommended that the infected culture be dis- carded and replaced with a mycoplasma-free cell culture [3]. However, if a culture is irreplaceable, it may be necessary to attempt one of the specialized techniques for eliminating myeoplasma. Methods of mycoplasma eradication include passage in athymic