Eradication of Mycoplasma Contaminations
Chapter 2 Eradication of Mycoplasma Contaminations Cord C. Uphoff and Hans G. Drexler Abstract Mycoplasma contaminations have a multitude of effects on the cultured cell lines that may influence the results of experiments or pollute bioactive substances used in human medicine. The elimination of mycoplasma contaminations of cell cultures has become a practical alternative to discarding and reestablishing important or irreplaceable cell lines. Different quinolones, tetracyclines, and pleuromutilins shown to have strong antimycoplasma properties are employed for the decontamination. We provide detailed protocols to assure eradication of mycoplasma, to prevent formation of resistant mycoplasma strains, and to cure heavily contaminated and damaged cells. To date, we have not detected any consistent and permanent alterations to eukaryotic cells either during or after the treatment. Key words: Antibiotic elimination, Cell lines, Mycoplasma
1. Introduction The use of human and animal cell lines for the examination of biological functions and for the production of bioactive substances requires rigorous quality control to exclude contamination with organisms (i.e., other eukaryotic cells, bacteria, and viruses). In this respect, mycoplasmas play an important but undesirable role, because a high portion (more than 20%) of the cell cultures arriving at our cell lines collection are contaminated with these wall-less bacteria. Mycoplasmas can have a multitude of effects on eukaryotic cells and can alter almost every cellular parameter, from proliferation via signaling pathways to virus susceptibility and production. Most striking are the effects resulting from the competition for nutrients that leads to the depletion of a number of essential nutrients. Consequentially, many downstream effects, such as altered
Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946, DOI 10.1007/978-1-62703-128-8_2, © Springer Science+Business Media, LLC 2013
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levels of protein, DNA, and RNA synthesis, and alterations of cellular metabolism and cell morphology, can be detected. Mycoplasmas do not gain energy by oxidative phosphorylation, but from the fermentative metabolism of diverse nutrients. This can lead to an alteration of the pH in the medium and to the production of metabolites that are toxic to the eukaryotic cells (e.g., NH3). The dependence of many mycoplasmas on cholesterols, sterols, and lipids can result in an alteration of the membrane composition. Other activation and suppression processes have also been described (e.g., lymphocyte activation, cytokine expression, induction of chromosomal aberrations, etc.). It has been noted that many experimentally analyzed parameters that were at first attributed to the eukaryotic cells were later ascribed to the contaminating mycoplasmas or were influenced by them. For example, mycoplasmas carry a uridine phosphorylase that can inactivate the artificial deoxynucleotide bromodeoxyuridine (BrdU). Cells with a thymidine kinase defect are commonly used for cell fusions and selected by the addition of BrdU. If mycoplasmas inactivate BrdU, the growing eukaryotic cells might appear to carry the enzyme deficiency and are misleadingly selected for cell fusions. Cell lines for virus propagation are also often affected by mycoplasma infections, leading to higher or lower titers of viruses (1). When an infected cell culture is detected, it should be autoclaved and discarded immediately and replaced by a mycoplasmafree culture. However, some cell lines are not replaceable because of unique characteristics of the cells or due to all the work that has been invested to manipulate those particular cells. A number of methods have been suggested to eradicate mycoplasmas from cell cultures. They comprise physical, chemical, immunological, and chemotherapeutic treatment. Some treatments are restricted to surfaces only (e.g., exposure to detergents), to eukaryotic cell-free solutions such as fetal bovine serum (FBS) (e.g., filtration through microfilters), and to specific mycoplasma species (e.g., culture with antimycoplasma antisera), are not practicable for a standard cell culture laboratory (e.g., in vivo passage of continuous cell lines through nude mice cell cloning), or are ineffective in eliminating the mycoplasmas quantitatively (e.g., heat treatment, exposure to complement) (2). That some mycoplasma species have the ability to penetrate the eukaryotic cell should also be considered. Mycoplasma fermentans is one of the main infecting mycoplasma species that can also enter eukaryotic cells. Thus, eliminating agents must also be active intracytoplasmically. Chemotherapeutic treatment can be efficiently employed using specific antibiotics. Because mycoplasmas possess no rigid cell walls and have a highly reduced metabolism, many of the commonly used antibiotics exhibit no effect on the viability of the mycoplasmas. They are naturally resistant to antibiotics targeting cell wall biosynthesis (e.g., penicillins), have an acquired resistance against
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other antibiotics that are often prophylactically used in cell culture (e.g., streptomycin), or the antibiotics are effective only at concentrations which are detrimental to the eukaryotic cells as well. Hence, the general use of antibiotics in cell culture is not recommended, except under special circumstances and then only for short durations. General use of antibiotics could lead to selection of drug-resistant organisms, to lapses in aseptic technique, and to delayed detection of low-level infection with either mycoplasmas or other bacteria (3). Three classes of antibiotics have been shown to be highly effective against mycoplasmas, both in human/veterinary medicine and in cell culture: tetracyclines, pleuromutilins, and quinolones. These antibiotics can be used at relatively low concentrations, with a negligible likelihood of resistance development, and, finally, with low or no effects on the eukaryotic cells. Tetracyclines and pleuromutilins inhibit protein synthesis by binding to the 30S and 50S ribosomal subunits, respectively (4). Quinolones inhibit the bacterial DNA gyrase which is essential for mycoplasma DNA replication. The risk of development of resistant clones is minimized by the application of antibiotics with different mechanisms of action, by sufficient treatment durations, and by constant concentrations of the antibiotics in the medium (5). Here, we describe the use of several antibiotics for the treatment of mycoplasmacontaminated cells, the rescue of heavily infected cultures, the salvage treatment of resistant cultures, and some pitfalls during and after the treatment.
2. Materials (See Note 1) 1. BM-Cyclin (Roche, Mannheim, Germany) contains the pleuromutilin tiamulin (BM-Cyclin 1) and the tetracycline minocycline (BM-Cyclin 2), both in lyophilized states. Dissolve the antibiotics in 10 mL sterile distilled water (dH2O), aliquot in 1-mL fractions, and store at −20°C. These stock solutions have concentrations of 2.5 mg/mL and 1.25 mg/mL, respectively. Repeated freezing and thawing of the solutions are not detrimental to the activity of the antibiotics. The dissolved solutions can be used at 1:250 dilutions in cell culture (at 10 mg/ mL and 5 mg/mL final concentration, respectively). 2. Plasmocin (InvivoGen, San Diego, CA) contains two antibiotics; one is active against protein synthesis of the bacteria, and one inhibits the DNA replication (gyrase inhibitor) (specific types of reagents not disclosed). The mixture is a ready-to-use solution and applied 1:1,000 in the cell culture (at 25 mg/mL final concentration).
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3. Ciprobay 100 (Bayer, Leverkusen, Germany) is a ready-to-use solution containing 2 mg/mL ciprofloxacin. It can be used 1:200 in cell culture (at 10 mg/mL final concentration). Onemilliliter aliquots should be taken sterilely from the bottle and stored at 4°C. Crystals form at 4°C and can be redissolved at room temperature. 4. Baytril (Bayer) contains 100 mg/mL of enrofloxacin and is diluted 1:100 with RPMI 1640 medium immediately prior to the treatment. The dilution should be prepared freshly for every antimycoplasma treatment. This solution is used as 1:40 final dilution in cell culture (at 25 mg/mL final concentration). 5. Mycoplasma Removal Agent (MRA, ICN, Eschwege, Germany) is a ready-to-use dilution containing 50 mg/mL of a 4-oxoquinolone-3-carboxylic acid derivative (specific type of reagent not disclosed). It is used in the treatment of cell cultures in 1:100 dilutions (at 0.5 mg/mL final concentration). 6. MycoZap (Lonza, Verviers, Belgium) is a combination of an antimicrobial peptide (MycoZap reagent 1) and an antibiotic (MycoZap reagent 2) (specific types of reagents not disclosed) that are employed consecutively. The solutions are ready-to-use. 7. Phosphate-buffered saline (PBS): 140 mM NaCl, 2.7 mM KCl, 7.2 mM Na2HPO4 × 12 H2O, 1.47 mM KH2PO4. Adjust to pH 7.2 and autoclave for 20 min at 121°C. 8. Cell culture media and supplements as appropriate and recommended for the particular cell lines.
3. Methods 3.1. Pretreatment Procedures
1. If no frozen reserve ampoules of the cell line are available, aliquots of the contaminated cell line should be stored frozen before treatment. Whenever possible, the ampoules should be kept isolated from noninfected cultures, either at −80°C for short time (over the complete curation time of 1–2 months) or, preferably, in liquid nitrogen in separate tanks (see Note 2). The ampoules should be marked properly as “mycoplasmapositive” to prevent a mix-up of ampoules containing cured or infected cells. After successful cure, these mycoplasma-positive ampoules should be removed and the cells destroyed by autoclaving. 2. Prepare the antibiotic working solutions freshly for every treatment and add the solution directly to the cell culture, not to the stored medium.
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3. The FBS concentration should be increased to 20% before, during, and for at least 2 weeks after the treatment to ensure optimal growth conditions, even if the cells grow well at lower concentrations. 3.2. Antibiotic Treatment
Mycoplasma infection often impairs the growth and viability of eukaryotic cells. After addition of the antibiotic, heavily infected cells might recover immediately and the viability of the culture might increase rapidly. However, in other cases, the delicate health of the cells is further aggravated by the exposure to the antibiotics. One reason might be the partial inhibition of mitochondrial respiration by the antibiotic(s). Even though the optimal concentrations of the antibiotics were determined in many trials, different cell types or cells under different infection conditions might behave differently upon treatment. Thus, in some instances, the cultures might be killed by the treatment (5). In these events, the treatment must be repeated with an aliquot that was stored frozen prior to the treatment. Even when no antibiotics are added to the medium, the cells might reach a crisis and die. To counteract the treatmentassociated harm, a few general suggestions should be followed to improve the culture conditions and to reduce the stress of infection and treatment on the eukaryotic cells (these rules are suitable for most cell lines, but some cell lines require special care which must be determined by the user): ●
Keep the concentration of the antibiotic constant during the treatment period; degradation of the antibiotic can be avoided by frequent complete exchange of the medium noting the following caveats.
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Culture the cells at a medium or higher cell density and keep this density almost constant during the treatment and for a few weeks after; a higher density of the cells demands a more frequent change of medium, which is commonly preferable to a relatively low cell density and long intervals between medium changes. However, some cell lines reportedly produce their own growth factors and, therefore, the medium should not be fully exchanged, depending on the cell line (see Note 3).
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Observe the culture daily under the inverted microscope to recognize quickly any alteration in general appearance, growth, or morphology, decrease in cell viability, detachment of cells, formation of granules, vacuoles, and so forth.
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In the case of deterioration of the cell culture, interrupt the treatment for a few days and let the cells recover (but this should only be the last resort); culture conditions should be changed immediately after recognition of the alterations, because if the cells are already beyond a certain degree of damage, it is usually difficult to reverse the progression of apoptosis.
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If possible, frequently detach slowly growing adherent cells in order to facilitate the exposure of all mycoplasmas to the antibiotic; the contaminants should not have the opportunity to survive in sanctuaries such as cell membrane pockets. It is similarly helpful to break up clumps of suspension cells by vigorous pipetting or using other reagents (e.g., trypsin, TrypLE Express (Invitrogen, Darmstadt, Germany], or Accutase (Sigma, Munich, Germany]).
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As antibiotics are light sensitive; protect cultures from the light, as much as possible.
Generally, there are three different approaches to the treatment of infected cell cultures: (1) the use of a single antibiotic compound (e.g., the quinolones), with basically the same procedure employed for each antibiotic of that group; (2) the simultaneous application of two different antibiotics (e.g., in the case of Plasmocin); and (3) the use of a combination therapy applying two antimycoplasma agents subsequently (e.g., MycoZap) or in alternating cycles (e.g., BM-Cyclin) (4) (see Fig. 1 and Note 4). The latter method is more time-consuming, but also highly effective, and avoids the possible interference of two antibiotics. For example, the action of the bactericidal quinolones depends on the proliferation of the cells, which is compromised by bacteriostatic agents, such as tetracyclines. We recommend using two of the three types of treatment in parallel or subsequently, if one method fails. A schematic overview of the procedure is given in Fig. 1; an exemplary representation of the treatment with BM-Cyclin is shown in Fig. 2. 3.2.1. Treatment with BM-Cyclin
1. Prepare a cell suspension (detach adherent cells, break up clumps by pipetting or using other methods) (see Note 5); determine the cell density and viability by trypan blue exclusion staining. Seed the cells at a medium density (see Note 6) in a 25 cm2 flask or one well of a 6- or 24-well culture plate with the appropriate fresh and rich culture medium (10 mL for the flask, and 4 mL and 2 mL for the wells, respectively). Add 4 mL of a 2.5 mg/mL solution BM-Cyclin 1 (tiamulin) per milliliter of medium. Incubate the cell culture for 2 days. 2. Remove all cell culture medium in flasks or wells containing adherent cells or after centrifugation of suspension cells. If applicable, dilute the cell cultures to a medium cell density. Add fresh medium and the same concentration of BM-Cyclin 1 as used in step 1. Incubate for another day. This procedure will keep the concentration of the antibiotic approximately constant over the 3-day application of tiamulin. 3. Remove the medium and wash the cells once with PBS to remove the residual antibiotic agent completely from the cells
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Mycoplasma-infected Cell Line
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1
Freeze Aliquots of the Infected Cell Line as Back-up
2
Treat Cell Line with Quinolone
Plasmocin
MycoZap
BM-Cyclin
Mycoplasma PCR Testing
Mycoplasma-positive
Mycoplasma-negative
Expand Cells, Freeze Master Stock, Store in Liquid Nitrogen, Discard Mycoplasma-positive Back-ups
Quinolone- / Plasmocin- / MycoZap-resistant BM-Cyclin-resistant
(1) BM-Cyclin Treatment (2) Treatment of Untreated Aliquot with another Quinolone / Plasmocin / MycoZap (3) Other Elimination Method
(1) Repeat Treatment of Untreated Aliquot with BM-Cyclin (2) Treatment of Untreated Aliquot with a Quinolone / Plasmocin
Fig. 1. Scheme for mycoplasma eradication. Different antibiotics can be used to treat mycoplasma-contaminated cell lines with a high rate of expected success. We recommend (1) cryopreservation of original mycoplasma-positive cells as backups and (2) splitting of the growing cells into different aliquots. These aliquots should be exposed singly to the various antibiotics. Posttreatment mycoplasma analysis and routine monitoring with a sensitive and reliable method (for example by PCR) are of utmost importance.
and loosely attached mycoplasmas. Seed the cells at the appropriate density (as described in step 1; see Note 6) and add 4 mL of the 1.25 mg/mL solution BM-Cyclin 2 (minocycline) per milliliter of medium. Incubate the culture for 2 days. 4. Remove the culture medium and substitute with fresh medium. Add the same concentration of BM-Cyclin 2 as used in step 3. Washing with PBS is not necessary at this step. Incubate the cell culture for 2 days to complete the 4-day minocycline treatment.
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Treatment W
0
W
4
6
W
W
MycoplasmaTesting
W
W
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Days
Antibiotic-free Medium 1
12
2
1
1 2
2
1
1 2
2
Medium with 10 µg/mL BM-Cyclin 1 or 5 µg/mL BM-Cyclin 2 Fig. 2. Treatment protocol for BM-Cyclin. Antibiotics are given on the days indicated by arrows. Cells are washed (indicated by W) with PBS prior to the cyclical change of antibiotics to avoid formation of resistant mycoplasmas due to low concentrations of the antibiotics. At the end of the decontamination period, cells are washed with PBS and suspended in antibioticfree medium. After a minimum of 2 weeks of posttreatment, the mycoplasma status of the cells is examined with sensitive and robust methods (for example by PCR).
5. After washing the cells with PBS, repeat steps 1–4 twice (three cycles of BM-Cyclin 1 and BM-Cyclin 2 altogether). Proceed with Subheading 3.3. 3.2.2. Treatment with Quinolones and Plasmocin
1. Prepare a cell suspension (detach adherent cells, break up clumps by pipetting or using other methods) (see Note 5); determine the cell density and viability by trypan blue exclusion staining. Seed the cells at a medium density (see Note 6) in a 25 cm2 flask or one well of a 6- or 24-well culture plate with the appropriate fresh and rich culture medium (10 mL for the flask, and 4 mL and 2 mL for the wells, respectively). Add one of the following antibiotics to the cell culture and incubate for 2 days: (a) 25 mL of a 1 mg/mL solution of enrofloxacin (Baytril) per milliliter of medium. (b) 10 mL of a 50 mg/mL solution of MRA per milliliter of medium. (c) 5 mL of a 2 mg/mL solution of ciprofloxacin (Ciprobay) per milliliter of medium. (d) 1 mL of a 25 mg/mL solution of Plasmocin per milliliter of medium. 2. Remove all cell culture medium in flasks or wells containing adherent cells or after centrifugation of suspension cells. If applicable, dilute the cell cultures to a medium cell density. Add fresh medium and the same concentration of the respective antibiotic as used in step 1. Incubate for another 2 days.
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3. If using enrofloxacin or MRA, repeat step 2 another two times (altogether an 8-day treatment). If using ciprofloxacin or Plasmocin, repeat step 2 five times (altogether a 14-day treatment). Proceed with Subheading 3.3. 3.2.3. Treatment with MycoZap
1. The activity of the antimicrobial peptide is influenced by the concentration of the FBS. Thus, the FBS concentration of the cell culture medium should not exceed 5% during the treatment with MycoZap reagent 1. Add 500 mL MycoZap reagent 1 to 4.5 mL cell culture medium supplemented with maximally 5% FBS. 2. Prepare a cell suspension (detach adherent cells, break up clumps by pipetting or using other methods) (see Note 5); determine the cell density and viability by trypan blue exclusion staining. Seed 5 × 105 cells in 4.5 mL cell culture medium supplemented with maximally 5% FBS in a 25 cm2 flask. Add 5 mL medium containing MycoZap reagent 1 prepared in step 1 and incubate the cells until the culture reaches a medium density, but at least for 2 days. 3. Remove the complete cell culture medium in the flask containing adherent cells or after centrifugation of suspension cells. If applicable, dilute the cell cultures to the medium cell density. Add 9.5 mL fresh medium (containing the FBS concentration appropriate for the cell culture) and 0.5 mL of MycoZap reagent 2. Incubate for 2 days. 4. Repeat step 3 another two times (altogether a 6-day treatment) (see Note 6). Proceed with Subheading 3.3.
3.3. Culture and Testing Post Treatment
1. After completion of the treatment, remove the antibiotics by washing the cells with PBS. Culture the cells in the same manner (enriched medium, higher cell concentration, etc.) as during the treatment period, but do not add any antibiotics. Even penicillin and streptomycin should not be added to the medium. Culture the cells for at least another 2 weeks. Even if initially the cells appear to be in good health after the treatment, the cells might go into a crisis after the treatment, especially following treatment with BM-Cyclin. The reason for this posttreatment crisis is not clear, but it might be a result of reduced activity of the mitochondria. Thus, the cell status should be frequently examined under the inverted microscope. 2. After passaging, test the cultures for mycoplasma contamination. If the cells are clean, freeze and store aliquots in liquid nitrogen. The cells in active culture have to be retested periodically to ensure continued freedom from mycoplasma contamination (see Note 7).
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3. After complete decontamination, expand the cells and freeze master stocks of the mycoplasma-free cell line and store them in liquid nitrogen to provide a continuous supply of clean cells. Discard the ampoules of mycoplasma-infected cells (see Fig. 1).
4. Notes 1. Store the antibiotics at the recommended concentrations, temperatures, and usually in the dark, and do not use them after the expiration date. Upon formation of precipitates, completely dissolve the crystals at room temperature in the dark before use. As the antibiotics are light sensitive, protect both the stock and working solutions from light. 2. Storage in liquid nitrogen might be a source of cell culture contamination with mycoplasmas. Indeed, mycoplasmas were shown to survive in liquid nitrogen even without cryopreservation. Once introduced into the nitrogen, mycoplasmas may persist in the tank for an indefinite time, not proliferating, but are able to contaminate cell cultures in leaky ampoules stored in the liquid phase of the nitrogen. Thus, storing the ampoules in the gaseous phase of the nitrogen is recommended to prevent contamination. Additionally, contaminated cell cultures and those of unknown status should be stored separately from noninfected cells, preferably in separate tanks to prevent a mix-up of contaminated and mycoplasma-free cell cultures. If this is not possible, store the ampoules at different locations within a tank. 3. Some cell lines are sensitive to a complete exchange of the medium. If the medium can only be exchanged partially, 50% of the antibiotic concentration should be added to the remaining conditioned medium that already contains the antibiotic, whereas 100% of the antibiotic concentration is added to the fresh medium. 4. It is advantageous to employ two types of treatments (BM-Cyclin and one of the quinolones or Plasmocin or MycoZap) in parallel, as usually at least one of the treatments is successful. In the rare event of resistance, cells of the untreated frozen backup aliquots can be thawed and treated again with another antibiotic. As MRA, ciprofloxacin, and enrofloxacin all belong to the group of quinolones, it is likely that the use of an alternative compound from the same group
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will produce the same end result (cure, resistance, or culture death). In the case of loss of the culture during or after the treatment, aliquots can be treated with quinolones, as these are usually better tolerated by the eukaryotic cells. We recommend using MycoZap which shows almost no effect on the growth parameters during the treatment procedure. This treatment or the use of MRA is also recommended when the cells are already in very bad condition prior to treatment and the number of available cells would suffice only for a single type of treatment. Sometimes, the cells recover rapidly after starting the treatment due to the immediate reduction of the mycoplasmas. 5. Adherent cells are detached by methods appropriate for the cell line being treated. It is important to break up all clumps and clusters and to detach cells from the surface of the culture vessels. Although the antibiotics are in solution and should be accessible to all parts of the cells, the membranes might be barriers that cannot be passed by the antibiotics. Mycoplasmas trapped within clumps of eukaryotic cells or even in cavities formed by the cell membrane of a single cell might be protected from the antibiotic. This is also the reason for the advice to keep the concentration of the antibiotic constantly high by frequently exchanging the medium. Some mycoplasma species were shown to penetrate the eukaryotic cells, which may be a source of resistance if the eukaryotic cell membrane is a barrier for the antibiotics. On the other hand, it was shown that specific antibiotics (e.g., ciprofloxacin) accumulate in the eukaryotic cells so that the concentration is higher inside the cells than in the extracellular environment. 6. Depending on the growth rate of the cell line, which might be severely altered by the antibiotic, the cell density should be reduced, kept constant, or even increased. If no data are available at all for a given cell culture or if the cell culture is in very poor condition, the cell density, growth rate, and viability should be recorded frequently and adjusted to improve the condition of the culture. 7. Applying the overly sensitive PCR for the detection of mycoplasma, we found that the treated cell cultures might show a weak false positive signal even after 2 weeks of posttreatment passaging. This is not necessarily the result of a resistance of the mycoplasmas and their regrowth, but might be caused by residual DNA in the culture medium. These cell cultures should not be discarded after being found positive, but should be retested after further culturing (see Chapter 1).
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References 1. Barile MF, Rottem S (1993) Mycoplasmas in cell culture. In: Kahane I, Adoni A (eds) Rapid diagnosis of mycoplasmas. Plenum, New York 2. Uphoff CC, Drexler HG (2010) Mycoplasma contamination of cell cultures. In: Flickinger M (ed) The encyclopedia of industrial biotechnology, vol 5. Wiley, New York 3. Uphoff CC, Drexler HG (2001) Prevention of mycoplasma contamination in leukemia-lymphoma cell lines. Hum Cell 14:244–247
4. Schmidt J, Erfle V (1984) Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines. Exp Cell Res 152: 565–570 5. Uphoff CC, Drexler HG (2002) Comparative antibiotic eradication of mycoplasma infections from continuous cell lines. In Vitro Cell Dev Biol Animal 38:86–89
1. Introduction The use of human and animal cell lines for the examination of biological functions and for the production of bioactive substances requires rigorous quality control to exclude contamination with organisms (i.e., other eukaryotic cells, bacteria, and viruses). In this respect, mycoplasmas play an important but undesirable role, because a high portion (more than 20%) of the cell cultures arriving at our cell lines collection are contaminated with these wall-less bacteria. Mycoplasmas can have a multitude of effects on eukaryotic cells and can alter almost every cellular parameter, from proliferation via signaling pathways to virus susceptibility and production. Most striking are the effects resulting from the competition for nutrients that leads to the depletion of a number of essential nutrients. Consequentially, many downstream effects, such as altered
Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946, DOI 10.1007/978-1-62703-128-8_2, © Springer Science+Business Media, LLC 2013
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levels of protein, DNA, and RNA synthesis, and alterations of cellular metabolism and cell morphology, can be detected. Mycoplasmas do not gain energy by oxidative phosphorylation, but from the fermentative metabolism of diverse nutrients. This can lead to an alteration of the pH in the medium and to the production of metabolites that are toxic to the eukaryotic cells (e.g., NH3). The dependence of many mycoplasmas on cholesterols, sterols, and lipids can result in an alteration of the membrane composition. Other activation and suppression processes have also been described (e.g., lymphocyte activation, cytokine expression, induction of chromosomal aberrations, etc.). It has been noted that many experimentally analyzed parameters that were at first attributed to the eukaryotic cells were later ascribed to the contaminating mycoplasmas or were influenced by them. For example, mycoplasmas carry a uridine phosphorylase that can inactivate the artificial deoxynucleotide bromodeoxyuridine (BrdU). Cells with a thymidine kinase defect are commonly used for cell fusions and selected by the addition of BrdU. If mycoplasmas inactivate BrdU, the growing eukaryotic cells might appear to carry the enzyme deficiency and are misleadingly selected for cell fusions. Cell lines for virus propagation are also often affected by mycoplasma infections, leading to higher or lower titers of viruses (1). When an infected cell culture is detected, it should be autoclaved and discarded immediately and replaced by a mycoplasmafree culture. However, some cell lines are not replaceable because of unique characteristics of the cells or due to all the work that has been invested to manipulate those particular cells. A number of methods have been suggested to eradicate mycoplasmas from cell cultures. They comprise physical, chemical, immunological, and chemotherapeutic treatment. Some treatments are restricted to surfaces only (e.g., exposure to detergents), to eukaryotic cell-free solutions such as fetal bovine serum (FBS) (e.g., filtration through microfilters), and to specific mycoplasma species (e.g., culture with antimycoplasma antisera), are not practicable for a standard cell culture laboratory (e.g., in vivo passage of continuous cell lines through nude mice cell cloning), or are ineffective in eliminating the mycoplasmas quantitatively (e.g., heat treatment, exposure to complement) (2). That some mycoplasma species have the ability to penetrate the eukaryotic cell should also be considered. Mycoplasma fermentans is one of the main infecting mycoplasma species that can also enter eukaryotic cells. Thus, eliminating agents must also be active intracytoplasmically. Chemotherapeutic treatment can be efficiently employed using specific antibiotics. Because mycoplasmas possess no rigid cell walls and have a highly reduced metabolism, many of the commonly used antibiotics exhibit no effect on the viability of the mycoplasmas. They are naturally resistant to antibiotics targeting cell wall biosynthesis (e.g., penicillins), have an acquired resistance against
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other antibiotics that are often prophylactically used in cell culture (e.g., streptomycin), or the antibiotics are effective only at concentrations which are detrimental to the eukaryotic cells as well. Hence, the general use of antibiotics in cell culture is not recommended, except under special circumstances and then only for short durations. General use of antibiotics could lead to selection of drug-resistant organisms, to lapses in aseptic technique, and to delayed detection of low-level infection with either mycoplasmas or other bacteria (3). Three classes of antibiotics have been shown to be highly effective against mycoplasmas, both in human/veterinary medicine and in cell culture: tetracyclines, pleuromutilins, and quinolones. These antibiotics can be used at relatively low concentrations, with a negligible likelihood of resistance development, and, finally, with low or no effects on the eukaryotic cells. Tetracyclines and pleuromutilins inhibit protein synthesis by binding to the 30S and 50S ribosomal subunits, respectively (4). Quinolones inhibit the bacterial DNA gyrase which is essential for mycoplasma DNA replication. The risk of development of resistant clones is minimized by the application of antibiotics with different mechanisms of action, by sufficient treatment durations, and by constant concentrations of the antibiotics in the medium (5). Here, we describe the use of several antibiotics for the treatment of mycoplasmacontaminated cells, the rescue of heavily infected cultures, the salvage treatment of resistant cultures, and some pitfalls during and after the treatment.
2. Materials (See Note 1) 1. BM-Cyclin (Roche, Mannheim, Germany) contains the pleuromutilin tiamulin (BM-Cyclin 1) and the tetracycline minocycline (BM-Cyclin 2), both in lyophilized states. Dissolve the antibiotics in 10 mL sterile distilled water (dH2O), aliquot in 1-mL fractions, and store at −20°C. These stock solutions have concentrations of 2.5 mg/mL and 1.25 mg/mL, respectively. Repeated freezing and thawing of the solutions are not detrimental to the activity of the antibiotics. The dissolved solutions can be used at 1:250 dilutions in cell culture (at 10 mg/ mL and 5 mg/mL final concentration, respectively). 2. Plasmocin (InvivoGen, San Diego, CA) contains two antibiotics; one is active against protein synthesis of the bacteria, and one inhibits the DNA replication (gyrase inhibitor) (specific types of reagents not disclosed). The mixture is a ready-to-use solution and applied 1:1,000 in the cell culture (at 25 mg/mL final concentration).
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3. Ciprobay 100 (Bayer, Leverkusen, Germany) is a ready-to-use solution containing 2 mg/mL ciprofloxacin. It can be used 1:200 in cell culture (at 10 mg/mL final concentration). Onemilliliter aliquots should be taken sterilely from the bottle and stored at 4°C. Crystals form at 4°C and can be redissolved at room temperature. 4. Baytril (Bayer) contains 100 mg/mL of enrofloxacin and is diluted 1:100 with RPMI 1640 medium immediately prior to the treatment. The dilution should be prepared freshly for every antimycoplasma treatment. This solution is used as 1:40 final dilution in cell culture (at 25 mg/mL final concentration). 5. Mycoplasma Removal Agent (MRA, ICN, Eschwege, Germany) is a ready-to-use dilution containing 50 mg/mL of a 4-oxoquinolone-3-carboxylic acid derivative (specific type of reagent not disclosed). It is used in the treatment of cell cultures in 1:100 dilutions (at 0.5 mg/mL final concentration). 6. MycoZap (Lonza, Verviers, Belgium) is a combination of an antimicrobial peptide (MycoZap reagent 1) and an antibiotic (MycoZap reagent 2) (specific types of reagents not disclosed) that are employed consecutively. The solutions are ready-to-use. 7. Phosphate-buffered saline (PBS): 140 mM NaCl, 2.7 mM KCl, 7.2 mM Na2HPO4 × 12 H2O, 1.47 mM KH2PO4. Adjust to pH 7.2 and autoclave for 20 min at 121°C. 8. Cell culture media and supplements as appropriate and recommended for the particular cell lines.
3. Methods 3.1. Pretreatment Procedures
1. If no frozen reserve ampoules of the cell line are available, aliquots of the contaminated cell line should be stored frozen before treatment. Whenever possible, the ampoules should be kept isolated from noninfected cultures, either at −80°C for short time (over the complete curation time of 1–2 months) or, preferably, in liquid nitrogen in separate tanks (see Note 2). The ampoules should be marked properly as “mycoplasmapositive” to prevent a mix-up of ampoules containing cured or infected cells. After successful cure, these mycoplasma-positive ampoules should be removed and the cells destroyed by autoclaving. 2. Prepare the antibiotic working solutions freshly for every treatment and add the solution directly to the cell culture, not to the stored medium.
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3. The FBS concentration should be increased to 20% before, during, and for at least 2 weeks after the treatment to ensure optimal growth conditions, even if the cells grow well at lower concentrations. 3.2. Antibiotic Treatment
Mycoplasma infection often impairs the growth and viability of eukaryotic cells. After addition of the antibiotic, heavily infected cells might recover immediately and the viability of the culture might increase rapidly. However, in other cases, the delicate health of the cells is further aggravated by the exposure to the antibiotics. One reason might be the partial inhibition of mitochondrial respiration by the antibiotic(s). Even though the optimal concentrations of the antibiotics were determined in many trials, different cell types or cells under different infection conditions might behave differently upon treatment. Thus, in some instances, the cultures might be killed by the treatment (5). In these events, the treatment must be repeated with an aliquot that was stored frozen prior to the treatment. Even when no antibiotics are added to the medium, the cells might reach a crisis and die. To counteract the treatmentassociated harm, a few general suggestions should be followed to improve the culture conditions and to reduce the stress of infection and treatment on the eukaryotic cells (these rules are suitable for most cell lines, but some cell lines require special care which must be determined by the user): ●
Keep the concentration of the antibiotic constant during the treatment period; degradation of the antibiotic can be avoided by frequent complete exchange of the medium noting the following caveats.
●
Culture the cells at a medium or higher cell density and keep this density almost constant during the treatment and for a few weeks after; a higher density of the cells demands a more frequent change of medium, which is commonly preferable to a relatively low cell density and long intervals between medium changes. However, some cell lines reportedly produce their own growth factors and, therefore, the medium should not be fully exchanged, depending on the cell line (see Note 3).
●
Observe the culture daily under the inverted microscope to recognize quickly any alteration in general appearance, growth, or morphology, decrease in cell viability, detachment of cells, formation of granules, vacuoles, and so forth.
●
In the case of deterioration of the cell culture, interrupt the treatment for a few days and let the cells recover (but this should only be the last resort); culture conditions should be changed immediately after recognition of the alterations, because if the cells are already beyond a certain degree of damage, it is usually difficult to reverse the progression of apoptosis.
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C.C. Uphoff and H.G. Drexler ●
If possible, frequently detach slowly growing adherent cells in order to facilitate the exposure of all mycoplasmas to the antibiotic; the contaminants should not have the opportunity to survive in sanctuaries such as cell membrane pockets. It is similarly helpful to break up clumps of suspension cells by vigorous pipetting or using other reagents (e.g., trypsin, TrypLE Express (Invitrogen, Darmstadt, Germany], or Accutase (Sigma, Munich, Germany]).
●
As antibiotics are light sensitive; protect cultures from the light, as much as possible.
Generally, there are three different approaches to the treatment of infected cell cultures: (1) the use of a single antibiotic compound (e.g., the quinolones), with basically the same procedure employed for each antibiotic of that group; (2) the simultaneous application of two different antibiotics (e.g., in the case of Plasmocin); and (3) the use of a combination therapy applying two antimycoplasma agents subsequently (e.g., MycoZap) or in alternating cycles (e.g., BM-Cyclin) (4) (see Fig. 1 and Note 4). The latter method is more time-consuming, but also highly effective, and avoids the possible interference of two antibiotics. For example, the action of the bactericidal quinolones depends on the proliferation of the cells, which is compromised by bacteriostatic agents, such as tetracyclines. We recommend using two of the three types of treatment in parallel or subsequently, if one method fails. A schematic overview of the procedure is given in Fig. 1; an exemplary representation of the treatment with BM-Cyclin is shown in Fig. 2. 3.2.1. Treatment with BM-Cyclin
1. Prepare a cell suspension (detach adherent cells, break up clumps by pipetting or using other methods) (see Note 5); determine the cell density and viability by trypan blue exclusion staining. Seed the cells at a medium density (see Note 6) in a 25 cm2 flask or one well of a 6- or 24-well culture plate with the appropriate fresh and rich culture medium (10 mL for the flask, and 4 mL and 2 mL for the wells, respectively). Add 4 mL of a 2.5 mg/mL solution BM-Cyclin 1 (tiamulin) per milliliter of medium. Incubate the cell culture for 2 days. 2. Remove all cell culture medium in flasks or wells containing adherent cells or after centrifugation of suspension cells. If applicable, dilute the cell cultures to a medium cell density. Add fresh medium and the same concentration of BM-Cyclin 1 as used in step 1. Incubate for another day. This procedure will keep the concentration of the antibiotic approximately constant over the 3-day application of tiamulin. 3. Remove the medium and wash the cells once with PBS to remove the residual antibiotic agent completely from the cells
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Mycoplasma-infected Cell Line
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1
Freeze Aliquots of the Infected Cell Line as Back-up
2
Treat Cell Line with Quinolone
Plasmocin
MycoZap
BM-Cyclin
Mycoplasma PCR Testing
Mycoplasma-positive
Mycoplasma-negative
Expand Cells, Freeze Master Stock, Store in Liquid Nitrogen, Discard Mycoplasma-positive Back-ups
Quinolone- / Plasmocin- / MycoZap-resistant BM-Cyclin-resistant
(1) BM-Cyclin Treatment (2) Treatment of Untreated Aliquot with another Quinolone / Plasmocin / MycoZap (3) Other Elimination Method
(1) Repeat Treatment of Untreated Aliquot with BM-Cyclin (2) Treatment of Untreated Aliquot with a Quinolone / Plasmocin
Fig. 1. Scheme for mycoplasma eradication. Different antibiotics can be used to treat mycoplasma-contaminated cell lines with a high rate of expected success. We recommend (1) cryopreservation of original mycoplasma-positive cells as backups and (2) splitting of the growing cells into different aliquots. These aliquots should be exposed singly to the various antibiotics. Posttreatment mycoplasma analysis and routine monitoring with a sensitive and reliable method (for example by PCR) are of utmost importance.
and loosely attached mycoplasmas. Seed the cells at the appropriate density (as described in step 1; see Note 6) and add 4 mL of the 1.25 mg/mL solution BM-Cyclin 2 (minocycline) per milliliter of medium. Incubate the culture for 2 days. 4. Remove the culture medium and substitute with fresh medium. Add the same concentration of BM-Cyclin 2 as used in step 3. Washing with PBS is not necessary at this step. Incubate the cell culture for 2 days to complete the 4-day minocycline treatment.
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C.C. Uphoff and H.G. Drexler Posttreatment Passaging
Treatment W
0
W
4
6
W
W
MycoplasmaTesting
W
W
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Days
Antibiotic-free Medium 1
12
2
1
1 2
2
1
1 2
2
Medium with 10 µg/mL BM-Cyclin 1 or 5 µg/mL BM-Cyclin 2 Fig. 2. Treatment protocol for BM-Cyclin. Antibiotics are given on the days indicated by arrows. Cells are washed (indicated by W) with PBS prior to the cyclical change of antibiotics to avoid formation of resistant mycoplasmas due to low concentrations of the antibiotics. At the end of the decontamination period, cells are washed with PBS and suspended in antibioticfree medium. After a minimum of 2 weeks of posttreatment, the mycoplasma status of the cells is examined with sensitive and robust methods (for example by PCR).
5. After washing the cells with PBS, repeat steps 1–4 twice (three cycles of BM-Cyclin 1 and BM-Cyclin 2 altogether). Proceed with Subheading 3.3. 3.2.2. Treatment with Quinolones and Plasmocin
1. Prepare a cell suspension (detach adherent cells, break up clumps by pipetting or using other methods) (see Note 5); determine the cell density and viability by trypan blue exclusion staining. Seed the cells at a medium density (see Note 6) in a 25 cm2 flask or one well of a 6- or 24-well culture plate with the appropriate fresh and rich culture medium (10 mL for the flask, and 4 mL and 2 mL for the wells, respectively). Add one of the following antibiotics to the cell culture and incubate for 2 days: (a) 25 mL of a 1 mg/mL solution of enrofloxacin (Baytril) per milliliter of medium. (b) 10 mL of a 50 mg/mL solution of MRA per milliliter of medium. (c) 5 mL of a 2 mg/mL solution of ciprofloxacin (Ciprobay) per milliliter of medium. (d) 1 mL of a 25 mg/mL solution of Plasmocin per milliliter of medium. 2. Remove all cell culture medium in flasks or wells containing adherent cells or after centrifugation of suspension cells. If applicable, dilute the cell cultures to a medium cell density. Add fresh medium and the same concentration of the respective antibiotic as used in step 1. Incubate for another 2 days.
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3. If using enrofloxacin or MRA, repeat step 2 another two times (altogether an 8-day treatment). If using ciprofloxacin or Plasmocin, repeat step 2 five times (altogether a 14-day treatment). Proceed with Subheading 3.3. 3.2.3. Treatment with MycoZap
1. The activity of the antimicrobial peptide is influenced by the concentration of the FBS. Thus, the FBS concentration of the cell culture medium should not exceed 5% during the treatment with MycoZap reagent 1. Add 500 mL MycoZap reagent 1 to 4.5 mL cell culture medium supplemented with maximally 5% FBS. 2. Prepare a cell suspension (detach adherent cells, break up clumps by pipetting or using other methods) (see Note 5); determine the cell density and viability by trypan blue exclusion staining. Seed 5 × 105 cells in 4.5 mL cell culture medium supplemented with maximally 5% FBS in a 25 cm2 flask. Add 5 mL medium containing MycoZap reagent 1 prepared in step 1 and incubate the cells until the culture reaches a medium density, but at least for 2 days. 3. Remove the complete cell culture medium in the flask containing adherent cells or after centrifugation of suspension cells. If applicable, dilute the cell cultures to the medium cell density. Add 9.5 mL fresh medium (containing the FBS concentration appropriate for the cell culture) and 0.5 mL of MycoZap reagent 2. Incubate for 2 days. 4. Repeat step 3 another two times (altogether a 6-day treatment) (see Note 6). Proceed with Subheading 3.3.
3.3. Culture and Testing Post Treatment
1. After completion of the treatment, remove the antibiotics by washing the cells with PBS. Culture the cells in the same manner (enriched medium, higher cell concentration, etc.) as during the treatment period, but do not add any antibiotics. Even penicillin and streptomycin should not be added to the medium. Culture the cells for at least another 2 weeks. Even if initially the cells appear to be in good health after the treatment, the cells might go into a crisis after the treatment, especially following treatment with BM-Cyclin. The reason for this posttreatment crisis is not clear, but it might be a result of reduced activity of the mitochondria. Thus, the cell status should be frequently examined under the inverted microscope. 2. After passaging, test the cultures for mycoplasma contamination. If the cells are clean, freeze and store aliquots in liquid nitrogen. The cells in active culture have to be retested periodically to ensure continued freedom from mycoplasma contamination (see Note 7).
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3. After complete decontamination, expand the cells and freeze master stocks of the mycoplasma-free cell line and store them in liquid nitrogen to provide a continuous supply of clean cells. Discard the ampoules of mycoplasma-infected cells (see Fig. 1).
4. Notes 1. Store the antibiotics at the recommended concentrations, temperatures, and usually in the dark, and do not use them after the expiration date. Upon formation of precipitates, completely dissolve the crystals at room temperature in the dark before use. As the antibiotics are light sensitive, protect both the stock and working solutions from light. 2. Storage in liquid nitrogen might be a source of cell culture contamination with mycoplasmas. Indeed, mycoplasmas were shown to survive in liquid nitrogen even without cryopreservation. Once introduced into the nitrogen, mycoplasmas may persist in the tank for an indefinite time, not proliferating, but are able to contaminate cell cultures in leaky ampoules stored in the liquid phase of the nitrogen. Thus, storing the ampoules in the gaseous phase of the nitrogen is recommended to prevent contamination. Additionally, contaminated cell cultures and those of unknown status should be stored separately from noninfected cells, preferably in separate tanks to prevent a mix-up of contaminated and mycoplasma-free cell cultures. If this is not possible, store the ampoules at different locations within a tank. 3. Some cell lines are sensitive to a complete exchange of the medium. If the medium can only be exchanged partially, 50% of the antibiotic concentration should be added to the remaining conditioned medium that already contains the antibiotic, whereas 100% of the antibiotic concentration is added to the fresh medium. 4. It is advantageous to employ two types of treatments (BM-Cyclin and one of the quinolones or Plasmocin or MycoZap) in parallel, as usually at least one of the treatments is successful. In the rare event of resistance, cells of the untreated frozen backup aliquots can be thawed and treated again with another antibiotic. As MRA, ciprofloxacin, and enrofloxacin all belong to the group of quinolones, it is likely that the use of an alternative compound from the same group
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will produce the same end result (cure, resistance, or culture death). In the case of loss of the culture during or after the treatment, aliquots can be treated with quinolones, as these are usually better tolerated by the eukaryotic cells. We recommend using MycoZap which shows almost no effect on the growth parameters during the treatment procedure. This treatment or the use of MRA is also recommended when the cells are already in very bad condition prior to treatment and the number of available cells would suffice only for a single type of treatment. Sometimes, the cells recover rapidly after starting the treatment due to the immediate reduction of the mycoplasmas. 5. Adherent cells are detached by methods appropriate for the cell line being treated. It is important to break up all clumps and clusters and to detach cells from the surface of the culture vessels. Although the antibiotics are in solution and should be accessible to all parts of the cells, the membranes might be barriers that cannot be passed by the antibiotics. Mycoplasmas trapped within clumps of eukaryotic cells or even in cavities formed by the cell membrane of a single cell might be protected from the antibiotic. This is also the reason for the advice to keep the concentration of the antibiotic constantly high by frequently exchanging the medium. Some mycoplasma species were shown to penetrate the eukaryotic cells, which may be a source of resistance if the eukaryotic cell membrane is a barrier for the antibiotics. On the other hand, it was shown that specific antibiotics (e.g., ciprofloxacin) accumulate in the eukaryotic cells so that the concentration is higher inside the cells than in the extracellular environment. 6. Depending on the growth rate of the cell line, which might be severely altered by the antibiotic, the cell density should be reduced, kept constant, or even increased. If no data are available at all for a given cell culture or if the cell culture is in very poor condition, the cell density, growth rate, and viability should be recorded frequently and adjusted to improve the condition of the culture. 7. Applying the overly sensitive PCR for the detection of mycoplasma, we found that the treated cell cultures might show a weak false positive signal even after 2 weeks of posttreatment passaging. This is not necessarily the result of a resistance of the mycoplasmas and their regrowth, but might be caused by residual DNA in the culture medium. These cell cultures should not be discarded after being found positive, but should be retested after further culturing (see Chapter 1).
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References 1. Barile MF, Rottem S (1993) Mycoplasmas in cell culture. In: Kahane I, Adoni A (eds) Rapid diagnosis of mycoplasmas. Plenum, New York 2. Uphoff CC, Drexler HG (2010) Mycoplasma contamination of cell cultures. In: Flickinger M (ed) The encyclopedia of industrial biotechnology, vol 5. Wiley, New York 3. Uphoff CC, Drexler HG (2001) Prevention of mycoplasma contamination in leukemia-lymphoma cell lines. Hum Cell 14:244–247
4. Schmidt J, Erfle V (1984) Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines. Exp Cell Res 152: 565–570 5. Uphoff CC, Drexler HG (2002) Comparative antibiotic eradication of mycoplasma infections from continuous cell lines. In Vitro Cell Dev Biol Animal 38:86–89
