Eradication of Mycoplasma Contaminations from Cell Cultures

Cord C. Uphoff1, Hans G. Drexler1

1 Leibniz‐Institute DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.5
DOI:  10.1002/0471142727.mb2805s106
Online Posting Date:  April, 2014
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Abstract

Mycoplasma contaminations have a multitude of effects on cultured cell lines that may influence the results of experiments or pollute bioactive substances isolated from the eukaryotic cells. The elimination of mycoplasma contaminations from cell cultures with antibiotics has been proven to be a practical alternative to discarding and re‐establishing important or irreplaceable cell lines. Different fluoroquinolones, tetracyclins, pleuromutilins, and macrolides shown to have strong anti‐mycoplasma properties are employed for the decontamination. These antibiotics are applied as single treatments, as combination treatment of two antibiotics in parallel or successively, or in combination with a surface‐active peptide to enhance the action of the antibiotic. The protocols in this unit allow eradication of mycoplasmas, prevention of the development of resistant mycoplasma strains, and potential cure of heavily contaminated and damaged cells. Consistent and permanent alterations to eukaryotic cells attributable to the treatment have not been demonstrated. Curr. Protoc. Mol. Biol. 106:28.5.1‐28.5.12. © 2014 by John Wiley & Sons, Inc.

Keywords: contamination; elimination; mycoplasma; antibiotic treatment

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Elimination of Mycoplasmas with Single Fluoroquinolones or Simultaneous Dual Antibiotic Treatment
  • Alternate Protocol 1: Elimination of Mycoplasmas with Dual Sequential Antibiotic Treatment Using BM‐Cyclin
  • Alternate Protocol 2: Elimination of Mycoplasmas with a Membrane‐Active Peptide and an Antibiotic (MycoZap)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Elimination of Mycoplasmas with Single Fluoroquinolones or Simultaneous Dual Antibiotic Treatment

  Materials
  • Cells
  • Cell culture medium and supplements (e.g., fetal bovine serum and dimethyl sulfoxide) as appropriate and recommended for the particular cell line
  • Phosphate‐buffered saline (PBS; ); autoclaved
  • Reagent to detach adherent cells as recommended for the particular cell line (e.g., trypsin, trypsin/EDTA, TrypLE Express, TNE buffer)
  • 0.4% Trypan blue solution (Sigma Aldrich)
  • Antibiotics including:
    • Baytril (see recipe)
    • Ciprobay 100 (see recipe)
    • MRA (see recipe)
    • Plasmocin (see recipe)
  • Liquid nitrogen
  • Hemacytometer, e.g., Neubauer counting chamber (Brand, Wertheim, Germany)
  • Centrifuge
  • Ampoules
  • Nunc freezing container or another freezing method
  • −80°C freezer or liquid nitrogen
  • 25‐cm2 flasks or 6‐ or 24‐well culture plates
  • 37°C incubator
  • Additional reagents and equipment for determining cell number and viability with a hemacytometer and trypan blue staining ( ) and testing for mycoplasma contamination (unit )

Alternate Protocol 1: Elimination of Mycoplasmas with Dual Sequential Antibiotic Treatment Using BM‐Cyclin

  Materials
  • Cell culture medium and supplements as appropriate and recommended for the particular cell lines
  • Fetal bovine serum (FBS)
  • Trypan blue solution
  • BM‐Cyclin (see recipe)
  • Reagents to detach adherent cells as recommended for the particular cell line (e.g., trypsin, trypsin/EDTA, TrypLE Express, TNE buffer)
  • Phosphate‐buffered saline (PBS; ); autoclaved
  • Liquid nitrogen
  • Hemacytometer, e.g., Neubauer counting chamber
  • Centrifuge
  • Ampoules
  • Nunc freezing container or another freezing method
  • −80°C freezer or liquid nitrogen
  • 37°C incubator
  • 25‐cm2 flasks or 6‐ or 24 well culture plate
  • Additional reagents and equipment for preparing a cell suspension ( ) and testing the cultures for mycoplasma contamination (unit )

Alternate Protocol 2: Elimination of Mycoplasmas with a Membrane‐Active Peptide and an Antibiotic (MycoZap)

  Materials
  • Cell culture medium for the particular cell lines
  • Fetal bovine serum (FBS)
  • Dimethyl sulfoxide (DMSO)
  • Trypan blue
  • Reagents to detach adherent cells as recommended for the particular cell line (e.g., trypsin, trypsin/EDTA, TrypLE Express, TNE buffer)
  • MycoZap reagent 1 and MycoZap reagent 2 (see recipe)
  • Hemacytometer, e.g., Neubauer counting chamber
  • Labeled ampoules
  • Nunc freezing containers, optional
  • −80°C freezer or liquid nitrogen
  • 25‐cm2 flasks
  • Additional reagents and equipment for preparing a cell suspension ( ) and testing the cultures for mycoplasma contamination (unit )
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Figures

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Literature Cited

Literature Cited
   Armstrong, S.E. , Mariano, J.A. , and Lundin, D.J. 2010. The scope of mycoplasma contamination within the biopharmaceutical industry. Biologicals 38:211‐213.
   Drexler, H.G. and Uphoff, C.C. 2002. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39:75‐90.
   Nir‐Paz, R. , Prévost, M.‐C. , Nicolas, P. , Blanchard, A. , and Wróblewski, H. 2002. Susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to membrane‐active peptides and enrofloxacin in human tissue cell cultures. Antimicrob. Agents Chemother. 46:1218‐1225.
   Rottem, S. and Barile, M.F. 1993. Beware of mycoplasmas. Tibtech 11:143‐151.
   Schmidt, J. and Erfle, V. 1984. Elimination of mycoplasma from cell cultures and establishment of mycoplasma‐free cell lines. Exp. Cell Res. 152:565‐570.
   Uphoff, C.C. and Drexler, H.G. 2001. Prevention of mycoplasma contamination in leukemia‐lymphoma cell lines. Human Cell 14:244‐247.
   Uphoff, C.C. and Drexler, H.G. 2002. Comparative antibiotic eradication of mycoplasma infections from continuous cell lines. In Vitro Cell. Dev. Biol. Anim. 38:86‐89.
   Uphoff, C.C. and Drexler, H.G. 2010. Contamination of cell cultures, mycoplasma. In The Encyclopedia of Industrial Biotechnology ( M. Flickinger , ed.) pp. 3611‐3630. John Wiley & Sons, Hoboken, New Jersey.
   Uphoff, C.C. , Denkmann, S.A. , and Drexler H.G. 2012. Treatment of mycoplasma contamination in cell cultures with Plasmocin. J. Biomed. Biotechnol. 2012:267678.
   Yavlovich, A. and Rottem, S. 2007. Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells. FEMS Microbiol. Lett. 266:156‐162.
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