Basic Cell Culture Protocols

1

Melisso. Testimonienza e frammenti La Nuova Italia A cura di Giovanni Reale Χρόνος: 1970 Γλώσσα: italian File: PDF, 13.00 MB

2

Frammenti La Nuova Italia Eraclito, Miroslav Marchovic Χρόνος: 1978 Γλώσσα: italian File: PDF, 11.68 MB

METHODS

IN

MOLECULAR BIOLOGY™

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:
http://www.springer.com/series/7651

Basic Cell Culture Protocols
Fourth Edition

Edited by

Cheryl D. Helgason
Experimental Therapeutics, BC Cancer Agency, Vancouver, BC, Canada

Cindy L. Miller
STEMCELL Technologies Inc., Vancouver, BC, Canada

Editors
Cheryl D. Helgason
Experimental Therapeutics
BC Cancer Agency
Vancouver, BC
Canada

Cindy L. Miller
STEMCELL Technologies Inc.,
Vancouver, BC
Canada

ISSN 1064-3745
ISSN 1940-6029 (electronic)
ISBN 978-1-62703-127-1
ISBN 978-1-62703-128-8 (eBook)
DOI 10.1007/978-1-62703-128-8
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2012950590
© Springer Science+Business Media, LLC 2013
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Preface
Tissue culture techniques were first developed at the beginning of the twentieth century
and have undergone dramatic changes and improvements since that time. They are invaluable tools for the exploration of numerous biological questions related both to cellular
processes and to the signaling mechanisms that regulate them. At some point in their
careers, virtually every scientist and technician, as well as many medical professionals,
regardless of their area of specialization has a need to utilize cell culture systems.
Our objective in preparing this book was to provide the novice cell culturist with
sufficient information to perform the basic techniques, to ensure the health and identity of
their cell lines, and to be able to isolate and culture specialized primary cell types. It was our
intent to generate a valuable resource book containing clear methodologies pertinent to
current areas of investigation, rather than attempting to educate cell culturists on specific
cell types or organ systems. We have thus included updates on several of the chapters from
the previous edition but have also added a significant number of new chapters to this
volume.
It is anticipated that many readers will have a solid background in the fundamentals of
anatomy, histology, and biochemistry but little or no experience in cell culture. We anticipate that this book will be a useful resource for technicians, graduate students, and postdoctoral fellows, as well as for the research leaders (both basic scientists and clinicians) and cell
culture experts moving toward the use of new model systems.
The chapters that follow provide step-by-step instructions for the isolation and growth
of various primary cell types. In addition, they illustrate the techniques required for defining
the properties of various types of cells as well as for cell differentiation.
We wish to extend our sincerest appreciation to all the contributors who willingly took
the time to share their expertise and knowledge and to the many individuals who assisted
us in the preparation of this book. Special thanks go to Dr. Carrie Peters and Jessica Ata
who assisted with the editing process and were instrumental in keeping everything organized and on track.
Vancouver, BC, Canada
Vancouver, BC, Canada

Cheryl D. Helgason
Cindy L. Miller

v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v
xi

1 Detection of Mycoplasma Contaminations . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cord C. Uphoff and Hans G. Drexler
2 Eradication of Mycoplasma Contaminations . . . . . . . . . . . . . . . . . . . . . . . . . .
Cord C. Uphoff and Hans G. Drexler
3 STR DNA Typing of Human Cell Lines: Detection of Intraand Interspecies Cross-Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Wilhelm G. Dirks and Hans G. Drexler
4 Classical and Molecular Cytogenetic Analysis. . . . . . . . . . . . . . . . . . . . . . . . . .
Roderick A.F. MacLeod and Hans G. Drexler
5 Fluorescent In Situ Hybridization of DNA Probes in the Interphase
and Metaphase Stages of the Cell Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Linda A. Cannizzaro
6 The Development of T Lymphocytes in Fetal Thymus Organ Culture . . . . . . .
Takeshi Nitta, Izumi Ohigashi, and Yousuke Takahama
7 Generation, Isolation, and Engraftment of In Vitro-Derived
Human T Cell Progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Génève Awong and Juan Carlos Zúñiga-Pflücker
8 In Vitro Generation of Human T Regulatory Cells: Generation, Culture,
and Analysis of FOXP3-Transduced T Cells. . . . . . . . . . . . . . . . . . . . . . . . . . .
Alicia N. McMurchy and Megan K. Levings
9 Simultaneous Cloning and Selection of Hybridomas and Transfected Cell
Lines in Semisolid Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bert Wognum and Tracy Lee
10 Isolation and Characterization of Mouse Side Population Cells . . . . . . . . . . . .
Aysegul V. Ergen, Mira Jeong, Kuanyin K. Lin, Grant A. Challen,
and Margaret A. Goodell
11 Stem Cell Identification by DyeCycle Violet Side Population Analysis . . . . . . .
William G. Telford
12 Isolation and Characterization of Cancer Stem Cells In Vitro . . . . . . . . . . . . .
Craig Gedye and Laurie Ailles
13 Ex Vivo Differentiation of Cord Blood Stem Cells into Megakaryocytes
and Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nicolas Pineault, Amélie Robert, Valérie Cortin, and Lucie Boyer

1

vii

15

27
39

61
85

103

115

133
151

163
181

205

viii

Contents

14 Generation and Characterization of Murine Alternatively
Activated Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Shelley B. Weisser, Keith W. McLarren, Etsushi Kuroda, and Laura M. Sly
15 Human Long-Term Culture Initiating Cell Assay . . . . . . . . . . . . . . . . . . . . . .
Min Liu, Cindy L. Miller, and Connie J. Eaves
16 Long-Term Culture-Initiating Cell Assay for Mouse Cells . . . . . . . . . . . . . . . .
Stefan Woehrer, Cindy L. Miller, and Connie J. Eaves
17 Colony Forming Cell Assays for Human Hematopoietic Progenitor Cells . . . .
Bert Wognum, Ning Yuan, Becky Lai, and Cindy L. Miller
18 Studying Leukocyte Recruitment Under Flow Conditions. . . . . . . . . . . . . . . .
Sean A. Parsons, Christophe Jurzinsky, Susan L. Cuvelier,
and Kamala D. Patel
19 Generation and Establishment of Murine Adherent Cell Lines. . . . . . . . . . . . .
Rouzanna Istvanffy and Robert A.J. Oostendorp
20 Isolation, Enumeration, and Expansion of Human Mesenchymal
Stem Cells in Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ravenska Wagey and Brenton Short
21 Isolation and Culture of Mesenchymal Stem Cells from Mouse
Compact Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Brenton Short and Ravenska Wagey
22 Generation of a Pool of Human Platelet Lysate and Efficient
Use in Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Katharina Schallmoser and Dirk Strunk
23 In Vitro Methods to Culture Primary Human Breast Epithelial Cells. . . . . . . .
Afshin Raouf and Yu Jia Sun
24 Human Prostate Epithelial Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Johng S. Rhim
25 Enzymatic Dissociation, Flow Cytometric Analysis, and Culture
of Normal Mouse Mammary Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Michael Prater, Mona Shehata, Christine J. Watson, and John Stingl
26 Isolation and Characterization of Human Hair Follicle Epithelial Cells . . . . . .
Keita Inoue and Kotaro Yoshimura
27 Cocultivation of Human Oral Keratinocytes and Human
Osteoblast-Like Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ricarda Glaum and Margit Wiedmann-Al-Ahmad
28 Isolation and Culture of Skeletal Muscle Myofibers as a Means to
Analyze Satellite Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Paul Keire, Andrew Shearer, Gabi Shefer, and Zipora Yablonka-Reuveni
29 Hepatic Differentiation of Embryonic Stem Cells by Murine Fetal Liver
Mesenchymal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Takamichi Ishii, Kentaro Yasuchika, and Iwao Ikai
30 Methods to Culture, Differentiate, and Characterize Neural Stem Cells
from the Adult and Embryonic Mouse Central Nervous System . . . . . . . . . . .
Sharon A. Louis, Carmen K.H. Mak, and Brent A. Reynolds

225
241
257
267
285

301

315

335

349
363
383

395
411

423

431

469

479

Contents

31 Feeder-Independent Culture Systems for Human Pluripotent Stem Cells. . . . .
Jennifer Moody
32 Formation of Embryoid Bodies from Human Pluripotent Stem Cells
Using AggreWell™ Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jennifer Antonchuk
33 Techniques in Embryoid Body Formation from Human Pluripotent
Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nirupama K. Shevde and Amber A. Mael
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

507

523

535
547

Contributors
LAURIE AILLES • Department of Medical Biophysics, Ontario Cancer Institute,
University of Toronto, Toronto, ON, Canada
JENNIFER ANTONCHUK • STEMCELL Technologies Inc, Vancouver, BC, Canada
GÉNÈVE AWONG • Department of Immunology, University of Toronto, Toronto,
ON, Canada
LUCIE BOYER • Département de Recherche et Développement, Héma-Québec,
Québec City, QC, Canada
LINDA A. CANNIZZARO • Department of Pathology, Montefiore Medical Center,
Albert Einstein College of Medicine, Bronx, NY, USA
GRANT A. CHALLEN • The Center for Cell and Gene Therapy, Baylor College of Medicine,
Houston, TX, USA
VALÉRIE CORTIN • Département de Recherche et Développement, Héma-Québec,
Québec City, QC, Canada
SUSAN L. CUVELIER • Calvin, Phoebe and Joan Snyder Institute for Infection,
Immunity and Inflammation, University of Calgary, Calgary, AB, Canada
WILHELM G. DIRKS • Department of Human and Animal Cell Lines, DSMZ, German
Collection of Microorganisms and Cell Cultures and German Biological Resource
Center, Braunschweig, Germany
HANS G. DREXLER • Department of Human and Animal Cell Lines, DSMZ, German
Collection of Microorganisms and Cell Cultures and German Biological Resource
Center, Braunschweig, Germany
CONNIE J. EAVES • Terry Fox Laboratory, British Columbia Cancer Agency,
Vancouver, BC, Canada
AYSEGUL V. ERGEN • The Center for Cell and Gene Therapy, Baylor College of Medicine,
Houston, TX, USA
CRAIG GEDYE • Ontario Cancer Institute, Toronto, ON, Canada
RICARDA GLAUM • Department of Oral and Maxillofacial Surgery,
Albert Ludwigs University, Freiburg, Germany
MARGARET A. GOODELL • Stem Cells and Regenerative Medicine Center, Baylor College
of Medicine, Houston, TX, USA
IWAO IKAI • Department of Surgery, Graduate School of Medicine, Kyoto University;
Department of Surgery, Kyoto Medical Center, National Hospital Organization,
Kyoto, Japan
KEITA INOUE • Department of Plastic and Reconstructive Surgery, Shizuoka Cancer
Center, Shizuoka, Japan
TAKAMICHI ISHII • Department of Surgery, Graduate School of Medicine,
Kyoto University, Kyoto, Japan
ROUZANNA ISTVANFFY • The Stem Cell Physiology Laboratory, Medizinische Klinik,
Technische Universität München, Munich, Germany

xi

xii

Contributors

MIRA JEONG • The Center for Cell and Gene Therapy, Baylor College of Medicine,
Houston, TX, USA; University of Science and Technology, Daejeon, South Korea
CHRISTOPHE JURZINSKY • Calvin, Phoebe and Joan Snyder Institute for Infection,
Immunity and Inflammation, University of Calgary, Calgary, AB, Canada
PAUL KEIRE • Department of Biological Structure, School of Medicine,
University of Washington, Seattle, WA, USA
ETSUSHI KURODA • Division of Gastroenterology, Department of Pediatrics,
BC Children’s Hospital and The Child and Family Research Institute,
University of British Columbia, Vancouver, BC, Canada
BECKY LAI • STEMCELL Technologies, Vancouver, BC, Canada
TRACY LEE • STEMCELL Technologies, Vancouver, BC, Canada
MEGAN K. LEVINGS • Department of Surgery, University of British Columbia,
Vancouver, BC, Canada
KUANYIN K. LIN • The Center for Cell and Gene Therapy, Baylor College of Medicine,
Houston, TX, USA
MIN LIU • Terry Fox Laboratory, Vancouver, BC, Canada; Private Chinese Culture
University, Taipei, Taiwan
SHARON A. LOUIS • STEMCELL Technologies, Vancouver, BC, Canada
RODERICK A.F. MACLEOD • Department of Human and Animal Cell Cultures,
DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany
AMBER A. MAEL • WiCell Research Institute, Madison, WI, USA
CARMEN K.H. MAK • STEMCELL Technologies, Vancouver, BC, Canada
KEITH W. MCLARREN • Division of Gastroenterology, Department of Pediatrics,
BC Children’s Hospital and The Child and Family Research Institute,
University of British Columbia, Vancouver, BC, Canada
ALICIA N. MCMURCHY • Department of Surgery, University of British Columbia,
Vancouver, BC, Canada
CINDY L. MILLER • STEMCELL Technologies, Vancouver, BC, Canada
JENNIFER MOODY • STEMCELL Technologies, Vancouver, BC, Canada
TAKESHI NITTA • Division of Experimental Immunology, Institute for Genome Research,
University of Tokushima, Tokushima, Japan
IZUMI OHIGASHI • Division of Experimental Immunology, Institute for Genome Research,
University of Tokushima, Tokushima, Japan
ROBERT A.J. OOSTENDORP • The Stem Cell Physiology Laboratory, Medizinische Klinik,
Technische Universität München, Munich, Germany
SEAN A. PARSONS • Calvin, Phoebe and Joan Snyder Institute for Infection,
Immunity and Inflammation, University of Calgary, Calgary, AB, Canada
KAMALA D. PATEL • Calvin, Phoebe and Joan Snyder Institute for Infection,
Immunity and Inflammation, University of Calgary, Calgary, AB, Canada
NICOLAS PINEAULT • Département de Recherche et Développement, Héma-Québec,
Université Laval, Québec City, QC, Canada
MICHAEL PRATER • Cancer Research, UK Cambridge Research Institute, Cambridge,
UK

Contributors

xiii

AFSHIN RAOUF • Department of Immunology, Faculty of Medicine, University of
Manitoba and Manitoba Institute of Cell Biology, CancerCare Manitoba,
Winnipeg, MB, Canada
BRENT A. REYNOLDS • Department of Neurosurgery, University of Florida, Gainesville,
FL, USA
JOHNG S. RHIM • Department of Defense, Center for Prostate Disease Research,
Rockville, MD, USA
AMÉLIE ROBERT • Département de Recherche et Développement, Héma-Québec,
Québec City, QC, Canada
KATHARINA SCHALLMOSER • Stem Cell Research Unit Graz and Clinic for Blood Group
Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria
ANDREW SHEARER • Department of Biological Structure, School of Medicine,
University of Washington, Seattle, WA, USA
GABI SHEFER • Department of Biological Structure, Faculty of Medicine,
University of Washington, Seattle, WA, USA
MONA SHEHATA • Cancer Research UK, Cambridge Research Institute, Cambridge,
UK
NIRUPAMA K. SHEVDE • WiCell Research Institute, Madison, WI, USA
BRENTON SHORT • STEMCELL Technologies, Vancouver, BC, Canada
LAURA M. SLY • Division of Gastroenterology, Department of Pediatrics, BC Children’s
Hospital and The Child and Family Research Institute, University of British Columbia,
Vancouver, BC, Canada
JOHN STINGL • Cancer Research UK, Cambridge Research Institute, Cambridge, UK
DIRK STRUNK • Stem Cell Research Unit and Department of Hematology,
Medical University of Graz, Graz, Austria
YU JIA SUN • Department of Immunology, Faculty of Medicine, University of Manitoba
and Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, MB,
Canada
YOUSUKE TAKAHAMA • Division of Experimental Immunology, Institute for Genome
Research, The University of Tokushima, Tokushima, Japan
WILLIAM G. TELFORD • Experimental Transplantation and Immunology Branch,
National Institutes of Health, Bethesda, MD, USA
CORD C. UPHOFF • Department of Human and Animal Cell Lines, DSMZ,
German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
RAVENSKA WAGEY • STEMCELL Technologies, VancouverBC, Canada
CHRISTINE J. WATSON • Department of Pathology, University of Cambridge,
Cambridge, UK
SHELLEY B. WEISSER • Division of Gastroenterology, Department of Pediatrics,
BC Children’s Hospital and The Child and Family Research Institute, University of
British Columbia, Vancouver, BC, Canada
MARGIT WIEDMANN-AL-AHMAD • Department of Oral and Maxillofacial Surgery,
Albert Ludwigs University, Freiburg, Germany
BERT WOGNUM • STEMCELL Technologies, Vancouver, BC, Canada
STEFAN WOEHRER • Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada;
Medical University of Vienna, Vienna, Austria

xiv

Contributors

ZIPORA YABLONKA-REUVENI • Department of Biological Structure, University of
Washington, Seattle, WA, USA
KENTARO YASUCHIKA • Department of Surgery, Graduate School of Medicine,
Kyoto University, Kyoto, Japan
KOTARO YOSHIMURA • Department of Plastic Surgery, University of Tokyo, Tokyo, Japan
NING YUAN • STEMCELL Technologies, Vancouver, BC, Canada
JUAN CARLOS ZUÑIGA-PFLÜCKER • Department of Immunology, University of Toronto,
Toronto, ON, Canada

Chapter 1
Detection of Mycoplasma Contaminations
Cord C. Uphoff and Hans G. Drexler
Abstract
Mycoplasma contamination of cell lines is one of the major problems in cell culture technology. The
specific, sensitive, and reliable detection of mycoplasma contamination is an important part of mycoplasma
control and should be an established method in every cell culture laboratory. New cell lines as well as cell
lines in continuous culture must be tested in regular intervals. The polymerase chain reaction (PCR) methodology offers a fast and sensitive technique to monitor all cultures in a laboratory and can also be used to
determine the contaminating species.
The described assay can be performed in less than 3 hours, including sample preparation, DNA extraction, PCR run, and analysis of the PCR products. Special emphasis is given to steps taken to avoid falsenegative results due to the presence of inhibitors of the Taq polymerase in the crude samples and the
interpretation of the results. The technique can also be adapted to the requirements of the pharmacopoeia.
Key words: Bacteria, Cell lines, Contamination, Mycoplasma, PCR

1. Introduction
1.1. Mycoplasma
Contaminations
of Cell Lines

Acute contaminations of cell lines are frequently observed in
routine cell culture and can often be attributed to improper
handling of the growing culture. These contaminations can usually be
detected by the turbidity evolving after a short incubation time or
by routine observation of the cell culture under the inverted
microscope. In addition to these obvious contaminations, other
hidden infections can occur consisting of mycoplasmas, viruses, or
cross-contaminations with other cell lines. Although known for
many years and despite the multitude of publications dealing with
mycoplasma infections of cell cultures, a high proportion of scientists are not aware of the potential contamination of cell cultures
with mycoplasmas. As seen in our cell repository, more than 20%
of the incoming cell lines are infected with mycoplasmas, and in

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_1, © Springer Science+Business Media, LLC 2013

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C.C. Uphoff and H.G. Drexler

most cases, the depositor was not aware of this. Whereas in the
early years of cell culture, bovine serum was one of the major
sources of infections, nowadays mycoplasmas seem to be mainly
transferred from one infected culture to another by using laboratory equipment, media, or reagents that came into contact with
infected cultures. This culture hopping is concordant with the
occurrence of cell cross-contaminations with a proved incidence
of 15% plus an estimated number of unknown cases (see STR
DNA Typing of Human Cell Lines: Detection of Intra- and
Interspecies Cross-contamination) and is substantiated by the
finding that either all or none of the cell cultures of a given laboratory are infected with the same mycoplasma strain. Thus, methods
for the detection, elimination (see Chapter 2), and prevention (1)
of mycoplasma contaminations should belong to the basic
panel of cell culture techniques applied.
The term “Mycoplasma” is commonly used as synonym for the
class of Mollicutes that represents a large group of highly specialized bacteria and which are all characterized by their lack of a rigid
cell wall. Mycoplasma is the largest genus within this class. Due to
their small size and flexibility, some of these bacteria are able to
pass through conventional microbiological filters (0.2 mm). Their
reduced metabolic abilities cause a relatively long generation time
which is often in the range of that of cell lines, and they do normally not overgrow or kill the eukaryotic cells. The extent of infection is highly diverse and depends on the mycoplasma species, cell
type, and culture conditions. Their influence on the biological
characteristics of the eukaryotic cells is manifold and almost every
experimental or production setting can be influenced.
The identification of infecting mycoplasmas shows that only a
limited number of about seven Mycoplasma and Acholeplasma species from human, swine, and bovine natural hosts occur predominantly in cell cultures, and no species specificity can be observed.
Additionally, a few mycoplasma species were shown to enter the
eukaryotic cells actively and to exist intracytoplasmically (2).
M. fermentans is one of those penetrating mycoplasma species.
Hence, sensitive methods need to be established and frequently
employed in every cell culture laboratory to detect mycoplasma
contaminations.
1.2. Mycoplasma
Detection

The biological diversity of mycoplasmas and their close adaptation
to the cell cultures render it very difficult to detect all contaminations in one general assay. A wide spectrum of approaches have
been proposed to detect mycoplasmas in cell cultures. Most of
these methods are lengthy, complex, and not applicable in routine
cell culture (e.g., electron microscopy, biochemical and radioactive
incorporation assays, etc.), or are restricted to specific groups of
mycoplasmas. Molecular biological methods were the first to be
able to detect all the different mycoplasma types in cell cultures,

1

Detection of Mycoplasma Contaminations

3

regardless of their biological properties, with relatively low effort
in terms of time and labor (3).
Polymerase chain reaction (PCR) provides a very sensitive and
specific option for the direct detection of mycoplasmas in cell cultures. PCR combines many of the features previously offered by
different assays: sensitivity, specificity, low expenditure of labor,
time, and cost, simplicity, objectivity of interpretation, reproducibility, and documentation of the results. On the other hand, a
number of indispensable control reactions have to be included in
the PCR assay to avoid false-negative or false-positive results.
A comparison of the PCR method with other well-established
assays (DNA/RNA hybridization, microbiological culture) showed
that the PCR assay is a very robust, efficient, and reliable method
for the detection of mycoplasmas (4). Another advantage is the
possibility to adapt the method to the requirements of the pharmacopoeia, which demand a detection limit of 10 cfu/mL for each of
the seven different mycoplasma species (5). Each laboratory must
individually validate the assay.
The choice of the primer sequences is one of the most crucial
decisions. Several primer sequences are published for single or
nested PCR (see Note 1) and with narrow or broad specificity for
mycoplasma or eubacteria species. In most cases, the 16S rDNA
sequences are used as target sequences, because this gene contains
regions with more or less conserved sequences. This gene also
offers the opportunity to perform a PCR with the 16 S rDNA or a
reverse transcriptase-PCR (RT-PCR) with the cDNA of the 16 S
rRNA (see Note 2) (6). Here, we describe the use of a mixture of
oligonucleotides for the specific detection of mycoplasmas. This
approach reduces significantly the generation of false positive
results due to contaminations of the solutions for the sample preparation and the PCR run and of other materials with airborne bacteria. Nevertheless, major emphasis should be placed on the
preparation of the template DNA, the amplification of positive and
negative control reactions, and the observance of general rules for
the preparation of PCR reactions. One of the main problems concerning PCR reactions with samples from cell cultures is the inhibition of the Taq polymerase by unspecified substances. To eliminate
those inhibitors, we strongly recommend that the sample DNA be
extracted and purified by conventional phenol–chloroform extraction or by the more convenient column- or matrix-binding extraction methods.
We compared the conventional hot start PCR method with a
real-time PCR employing SYBR green DNA-binding dye and
found no significant advantage of the real-time PCR over the conventional PCR method. In contrast, due to the short amplification
cycles of the conventional PCR and the necessity to perform a
denaturation curve in the real-time PCR assay, the PCR method
presented here is faster and similarly convenient.

4

C.C. Uphoff and H.G. Drexler

To confirm the error-free preparation of the sample and PCR
run, appropriate control reactions have to be included in the PCR.
These comprise internal control DNA for every sample reaction
and, in parallel, positive and negative as well as water control reactions. The internal control consists of a DNA fragment with the
same primer sequences for amplification, but is of a different size
than the amplicon of mycoplasma-contaminated samples. This
control DNA is added to the PCR mixture in a previously determined limiting dilution to demonstrate the sensitivity of the PCR
reaction. In this chapter, detailed protocols are provided to establish the PCR method for the monitoring of mycoplasma contaminations in any laboratory.

2. Materials
1. Phosphate-buffered saline (PBS): 140 mM NaCl, 2.7 mM
KCl, 7.2 mM Na2HPO4 × 12 H2O, 1.47 mM KH2PO4, pH
7.2. Autoclave for 20 min at 121°C to sterilize the solution.
2. 50× Tris–acetic acid–EDTA (TAE): 2 M Tris base, 5.71% glacial acetic acetate (v/v), 100 mM EDTA. Adjust to pH of
approx. 8.5.
3. DNA extraction and purification system (e.g., phenol/chloroform extraction and ethanol precipitation, or DNA extraction
kits applying DNA binding matrices).
4. GeneAmp 9700 thermal
Weiterstadt, Germany).

cycler

(Applied

Biosystems,

5. Platinum Taq DNA polymerase, hot start, 5 U/mL (Invitrogen,
Karlsruhe, Germany), including 10× PCR buffer provided by
the manufacturer.
6. 50 mM MgCl2.
7. 6× loading buffer: 0.09% (w/v) bromophenol blue, 0.09%
(w/v) xylene cyanol FF, 60% glycerol (v/v), 60 mM EDTA.
8. Primers (any supplier) (see Note 3):
5¢ primers (Myco-5¢):
cgc ctg agt agt acg twc gc
tgc ctg rgt agt aca ttc gc
crc ctg agt agt atg ctc gc
cgc ctg ggt agt aca ttc gc
3¢ primers (Myco-3¢):
gcg gtg tgt aca ara ccc ga
gcg gtg tgt aca aac ccc ga

1

Detection of Mycoplasma Contaminations

5

(r = mixture of g and a; w = mixture of t and a)
Stock solutions: 100 mM in dH2O, stored frozen at −20°C.
Working solutions: Mixture of forward primers at 5 mM each
(Myco-5¢) and mixture of reverse primers at 5 mM each (Myco3¢) in dH2O, aliquoted in small amounts (i.e., 25–50 mL aliquots), and stored frozen at −20°C.
9. Mycoplasma PCR internal control DNA (4): Plasmid containing an insert producing a longer PCR product than the mycoplasma wild-type PCR product. The plasmid can be obtained
from the DSMZ (German Collection of Microorganisms and
Cell Cultures, Braunschweig, Germany). Before use, a limiting
dilution should be determined experimentally by performing a
PCR with a dilution series of the internal control DNA (see
Note 4).
10. Mycoplasma-positive control DNA: A tenfold dilution of any
mycoplasma-positive sample prepared as described in
Subheading 3.1, or obtained from the DSMZ.
11. 5 mM deoxy-nucleotide triphosphate mixture (dNTP-mix) is
prepared from the nucleotides of the 100 mM dNTP set
(Invitrogen, Karlsruhe, Germany) by diluting each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate
(dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP) 1:5 with dH2O and combining
the same volumes (e.g., 100 mL each). The resulting working
solution with each nucleotide at a concentration of 5 mM is
then aliquoted in small amounts (i.e., 25–50 mL aliquots) and
stored at −20°C.
12. 1.3% Agarose-TAE gel (7).
13. Optional: Restriction enzymes AspI, HaeIII, HpaII, XbaI
including the 10× restriction enzyme buffers recommended by
the manufacturer.

3. Methods
The following subsections describe the sample collection, extraction of the DNA, setting up and performing the PCR reaction,
interpretation of the results, and, in addition, the identification of
the mycoplasma species. These techniques can also be used to
detect mycoplasma contamination in culture media or other supplements (see Note 5).
Every incoming cell culture should be kept in quarantine until
mycoplasma detection assays are completed and the infection status
is clearly determined. Positive cultures should either be discarded

6

C.C. Uphoff and H.G. Drexler

and replaced by clean cultures or cured with specific antibiotics
(see Chapter 2). Only definitely clean cultures should be used for
research experiments and for the production of biologically active
pharmaceuticals. Additionally, stringent rules for the prevention of
further mycoplasma contamination of cell cultures should be
strictly followed (1).
3.1. Sample Collection
and Preparation
of DNA

1. Prior to collecting the samples, the cell line to be tested for
mycoplasma contamination should be in continuous culture
for several days and without any antibiotics (even penicillin and
streptomycin) or for at least 2 weeks after thawing. This should
assure that the titer of the mycoplasmas in the supernatant is
above the detection level of the PCR assay.
2. Take one milliliter of the supernatant of adherently growing
cells or of cultures with settled suspension cells (by placing the
cell culture vessel in an upright position for approximately
30–60 min) for the analysis. By collecting the samples in this
way, some viable and/or dead eukaryotic cells are always present
in the samples. This is intended as some mycoplasma strains predominantly adhere to the eukaryotic cells or even invade them.
On the other hand, only a limited number of eukaryotic cells is
desired to prevent an excess of eukaryotic DNA. Thus, it is also
not necessary to centrifuge the sample to eliminate the eukaryotic cells. The crude cell culture supernatants can be stored at
4°C for a few days or frozen at −20°C for several weeks. After
thawing, the samples should be further processed immediately.
3. Centrifuge the cell culture suspension at 13,000 × g for 5 min.
Resuspend the pellet in 1 mL PBS by vortexing.
4. Centrifuge the suspension again and wash one more time with
PBS as described in step 3.
5. After centrifugation, resusupend the pellet in 100 mL PBS by
vortexing and heat to 95°C for 15 min.
6. Immediately after lysing the cells, extract and purify the DNA
by standard phenol/chloroform extraction and ethanol precipitation (7) or other DNA isolation methods (see Note 6).

3.2. PCR

The amplification procedure and the parameters described here are
optimized for the application in thin-walled 0.2-mL reaction tubes
in an Applied Biosystems GeneAmp 9700 thermal cycler. An
adjustment to any other equipment might be necessary (see Note
7). Amplified positive samples contain high amounts of target
DNA. Thus, established rules to avoid DNA carryover should be
strictly followed: (1) the places where the DNA is extracted, the
PCR reaction is set up, and the gel is run after the PCR should be
separated from each other; (2) all reagents should be stored in
small aliquots to provide a constant source of uncontaminated

1

Detection of Mycoplasma Contaminations

7

reagents; (3) avoid reamplifications; (4) reserve pipettes, tips, and
tubes for their use in the PCR only and irradiate the pipettes frequently by ultraviolet (UV) light; (5) the succession of the PCR
setup described below should be followed strictly; (6) wear gloves
during the whole sample preparation and PCR setup; and (7)
include the appropriate control reactions, such as internal, positive,
negative, and the water control reaction.
1. For each sample to be tested, set up one reaction with the sample only and another parallel reaction also containing the internal control DNA (to confirm the accuracy of the PCR run):
Sample only

Sample plus internal
control DNA

dNTPs

1 mL

1 mL

Myco-5¢

0.5 mL

0.5 mL

Myco-3¢

0.5 mL

0.5 mL

10× PCR buffer

2.5 mL

2.5 mL

MgCl2

1 mL

1 mL

dH2O

18.3 mL

17.3 mL

Platinum Taq polymerase

0.2 mL

0.2 mL

Internal control DNA

–

1 mL

When testing several samples, pre-master mixtures can be
prepared. For the reaction without internal control DNA two
reactions should be added (for the positive and the water control reactions) and for the reactions with the internal control
DNA two reactions should be added (for the positive and the
negative control reaction) (see Notes 4 and 8). For both premaster mixtures, also add the amounts for one additional reaction to have a surplus for pipetting variations.
2. Transfer 24 mL of each of the pre-master mixtures to 0.2 mL
PCR reaction tubes and add 1 mL dH2O to the water control
reaction and the internal control only reaction.
3. Set aside all reagents used for the preparation of the master
mixtures and take out the samples of DNA to be tested and the
positive control DNA. Do not handle the reagents and samples
simultaneously. For each DNA sample, and for the positive
control DNA, add 1 mL of the DNA preparation to a reaction
tube that contains no internal control DNA and another 1 mL
to a tube containing the internal control DNA.
4. Transfer the reaction tubes to the thermal cycler and start a
one-step cycle at 96°C for 2 min to activate the hot start Taq
polymerase.

8

C.C. Uphoff and H.G. Drexler

5. After this initial cycle, perform 35 thermal cycles with the
following parameters:
Cycle step 1: 4 s at 95°C.
Cycle step 2: 8 s at 65°C.
Cycle step 3: 16 s at 72°C plus 1 s of extension time during
each cycle.
6. Finish the reaction with a final amplification step at 72°C for
7 min, and then cool the samples down to room temperature.
7. Prepare a 1.3% agarose-TAE gel containing either 0.3 mg of
ethidium bromide per mL or with another nucleic acid gel
stain. Submerge the gel in 1× TAE and add 12 mL of the
amplification product (10 mL reaction mixtures plus 2 mL of 6×
loading buffer) to each well. Run the gel at 10 V/cm.
8. Visualize the specific products on a suitable UV screen and
document the result.
3.3. Interpretation
of Results

Figure 1 shows a representative ethidium bromide-stained gel with
some samples that produce the following results:
–

Ideally, all samples containing the internal control DNA show
a band at 986 bp. This band might be more or less bright, but
the band should be visible even if no other bands are amplified
(see Note 9). If the band is not visible, the reaction may have
been contaminated with Taq polymerase inhibitors from the
cell culture that were not removed by the DNA extraction
method. For example, melanin is a potent Taq polymerase
inhibitor that occurs in melanoma cell cultures and cannot be
eliminated by any DNA extraction method. In such a case,
bovine serum albumin (BSA) can be added to the sample at
high concentrations to overcome the inhibition. However,
usually it is sufficient to repeat the PCR run with the same
DNA solution as used previously. It is not necessary to collect
a new sample from the cell culture. If the second run also does
not show a band for the sample and internal control, the whole
procedure should be repeated.

–

Mycoplasma-positive samples show a band at 502–520 bp,
depending on the mycoplasma species. In the case of a
Acholeplasma laidlawii contamination, and when the DSMZ
internal control DNA is applied, a third band might be visible
between the internal control band and the mycoplasma-specific
band. This is formed by cross-hybridization of the complementary sequences of the single-stranded long internal control
DNA and the shorter, single-stranded wild-type mycoplasma
DNA form.

–

Contamination of reagents with mycoplasma-specific DNA or
PCR products is revealed by a band in the water control and/
or in the negative control sample.

1

Detection of Mycoplasma Contaminations

9

Fig. 1. PCR analysis of mycoplasma status in cell lines. Shown is an ethidium bromide-stained gel containing the reaction
products following PCR amplification with the primer mix listed in Subheading 2. Products of about 510 bp are obtained;
the differences in length reflect the sequence variation between different mycoplasma species. Shown are various examples of mycoplasma-negative and -positive cell lines. Two paired PCR reactions were performed: one PCR reaction contained an aliquot of the sample only (a) and the second reaction contained the sample under study plus the control DNA as
internal standard (b). Cell cultures A, C, and E are mycoplasma positive; cell culture B is mycoplasma negative. The result
of cell culture D is not evaluable because the internal control was not amplified and no other mycoplasma-specific band
appeared in the gel. In this case, the analysis needs to be repeated. Cell line C 2 weeks post antibiotic treatment shows a
weak but distinctive band in the reaction without internal control. This band results from residual DNA in the medium,
because after a further 2 weeks of culture no contamination was detected.

–

3.4. Identification of
Mycoplasma Species

Weak mycoplasma-specific bands can occur after treatment of
infected cell cultures with anti-mycoplasma reagents for the
elimination of mycoplasma or when other antibiotics are used
routinely. In these cases the positive reaction might be due to
residual DNA derived either from dead mycoplasma cells in
the culture medium or from viable mycoplasma cells which are
present at a very low titer. Therefore, special caution should be
taken when cell cultures are tested that were treated with antibiotics. Prior to PCR testing, cell cultures should be cultured
for at least 2–3 weeks without antibiotics or retested at frequent intervals to demonstrate either a decrease or an increase
of mycoplasma.

Although the method described is sufficient to detect mycoplasma
contaminations, it might be of advantage to know the infecting
mycoplasma species, e.g., in efforts to determine the source of a
contamination. This PCR allows the identification of the mycoplasma species most commonly infecting cell cultures by modified

10

C.C. Uphoff and H.G. Drexler
Xba I

M. arginini – M. hominis – M. hyorhinis – M. orale
~265 bp / ~253 bp

A. laidlawii – M. bovis – M. fermentans

Hpa II

Hpa II

M. arginini – M. hominis – M. hyorhinis

M. orale
288 bp / 230 bp

A. laidlawii – M. bovis

Asp I

M. hyorhinis
303 bp / 213 bp

M. fermentans
357 bp / 111 bp / 48 bp

Asp I

M. arginini – M. hominis

M. bovis
303 bp / 213 bp

A. laidlawii

Hae III

M. hominis
336 bp / 180 bp

M. arginini

digested
not digested

Fig. 2. Flowchart for the identification of the mycoplasma species. Digesting aliquots of the amplified PCR product with the
indicated restriction enzymes will result in undigested (solid lines ) or digested (dashed lines ) fragments of the sizes mentioned below the species names.

restriction fragment length polymorphism analysis. In case of a
contamination detected by PCR, the PCR reaction is repeated in a
50 mL volume without the internal control DNA to amplify only
the wild-type mycoplasma-specific PCR fragment. Per reaction,
8 mL of the amplified DNA is taken directly from the PCR reaction
and is digested in parallel reactions with the restriction endonucleases Asp I, Hae III, Hpa II, and Xba I by the addition of 1 mL
of the appropriate 10× restriction enzyme buffer and 1 mL of the
restriction enzyme. The mycoplasma species can be determined
directly by the restriction pattern (see Fig. 2). This analysis only
identifies those mycoplasma species which most often (>98%) occur
in cell cultures, and is not suitable for the global identification of
all types of mycoplasma species. Cell culture infections are commonly restricted to the mycoplasma species listed in Fig. 2.

4. Notes
1. Originally, the described method was designed as nested PCR
(8). Here, the second round of PCR was omitted, because
in standard applications no significant differences in the
results were observed between only one round of PCR and

1

Detection of Mycoplasma Contaminations

11

nested PCR. Mycoplasma-positive cell cultures were detected
as positive in the first round of PCR and negative samples
were consistently negative employing nested PCR.
Furthermore, applying a nested PCR increases the risk of
transmission of first-round PCR products to the reagents
used in the second amplification and possibly to those shared
with the first round.
2. In this protocol, genomic DNA is applied for the PCR reaction. As the primers hybridize to the 16S rRNA, an RT-PCR
can also be performed after extracting RNA and preparation of
cDNA. Theoretically, RT-PCR should increase the sensitivity
of the assay, because the number of rRNA molecules per organism is much higher than the number of the coding genes.
Nevertheless, we found no significant increase in sensitivity
employing cDNA, and thus the additional labor, time, and
costs required for RT-PCR are unwarranted.
3. The primers can be prepared using a degenerated code to
incorporate two different nucleotides at one site to form a
mixture of two primers. When the forward or reverse oligonucleotides are mixed and aliquoted for use in the PCR reaction, it must be taken into account that the molarities of the
oligonucleotides with mixed bases are reduced by 50%. The
primer solutions should be aliquoted into small portions
(i.e., 25 mL aliquots) and stored frozen at −20°C to avoid
multiple freeze-thawing cycles and to minimize contamination risks.
4. The limiting dilution of the internal control DNA can be used
for 2 or 3 months at most when stored at 4°C. After this time,
the amplification of the internal control DNA might fail even
when no inhibitors are present in the reaction, because the
DNA concentration may be reduced due to degradation or
adherence to the plastic tube.
5. To use this PCR method to test cell culture media or supplements expected to have low titers of mycoplasmas (e.g., fetal
bovine serum [FBS]), a larger quantity of the sample can be
centrifuged. The pellet is further processed as described in this
protocol without adjusting the reagent amounts to the higher
initial sample size.
6. We do not recommend using the crude lysate of a sample for
the PCR reaction as described in some publications, because it
often contains inhibitors of Taq polymerase and may lead to
false negative results. For convenience and speed of the assay
we use commercially available DNA extraction/purification
kits based on binding of the DNA to matrices and subsequent
elution of the DNA. We tested normal phenol/chloroform
extraction and ethanol precipitation, the High Pure PCR

12

C.C. Uphoff and H.G. Drexler

Template Preparation Kit from Roche (Mannheim, Germany),
the Invisorb DNA/RNA Virus Kit from Invitek (Berlin,
Germany), the Wizard DNA Clean-Up System (for plasmid
purification) from Promega (Mannheim, Germany), and the
Epicentre MasterPure Complete DNA & RNA Purification Kit
(Biozym, Hessisch Oldendorf, Germany). Following the recommendations of the manufacturers, the amplifications of the
mycoplasma sequences were all similar when the same amounts
were used for the elution or resuspension. For screening many
samples, the Wizard system works very well with the vacuum
manifold.
7. The use of thermal cyclers other than the GeneAmp 9700
might require some modifications in the amplification parameters (e.g., duration of the cycling steps, which are short in
comparison to other applications). Magnesium, primer, or
dNTP concentrations might also need to be altered. The same
may be true if another Taq polymerase is used, either polymerases from different suppliers or different kinds of Taq polymerase. Although the described PCR method is very robust,
we found that the type of Taq polymerase and the appropriate
buffer system have a major impact on the performance and
sensitivity of the PCR.
8. Using the internal control DNA, the described PCR method
is competitive only for the group of mycoplasma species that
carry primer sequences identical to the one from which the
internal control DNA was prepared. Other primer sequences
are not used up in the PCR reaction because of mismatches.
Usually, one reaction per sample is sufficient to detect mycoplasma in long-term infected cell cultures. But to avoid the
risk of performing a competitive reaction and of decreasing
the sensitivity of the PCR reaction (e.g., after anti-mycoplasma treatment or for the testing of cell culture reagents),
two separate reactions are performed: (1) without internal
control DNA to make all reagents available for the amplification
of the specific product and (2) including the internal control
DNA to demonstrate the integrity of the PCR reaction (see
Fig. 1).
9. Heavily infected cell cultures might show the mycoplasmaspecific band, whereas the internal control is not visible. In this
case the mycoplasma target DNA suppresses the internal control which is present in the reaction mixture at much lower
concentrations. The reaction is classified as mycoplasma positive (see Fig. 1).

1

Detection of Mycoplasma Contaminations

13

References
1. Uphoff CC, Drexler HG (2001) Prevention of
mycoplasma contamination in leukemia-lymphoma cell lines. Hum Cell 14:244–247
2. Drexler HG, Uphoff CC (2002) Mycoplasma
contamination of cell cultures: incidence,
sources, effects, detection, elimination, prevention. Cytotechnology 39:23–38
3. Uphoff CC, Drexler HG (2010) Contamination
of cell cultures, mycoplasma. In: Flickinger M
(ed) The encyclopedia of industrial biotechnology. Wiley, New York
4. Uphoff CC, Drexler HG (2002) Comparative
PCR analysis for detection of mycoplasma
infections in continuous cell lines. In Vitro Cell
Dev Biol Animal 38:79–85

5. European Pharmacopoeia (2008) General
chapter 2.6.7., Mycoplasmas, suppl. 5.6.
Council of Europe, Strasbourg, France
6. Uphoff CC, Drexler HG (1999) Detection of
mycoplasma contamination in cell cultures by
PCR analysis. Hum Cell 12:229–236
7. Sambrook J, Fritsch EF, Maniatis T (eds)
(1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY
8. Hopert A, Uphoff CC, Wirth M, Hauser H,
Drexler HG (1993) Specificity and sensitivity
of polymerase chain reaction (PCR) in comparison with other methods for the detection
of mycoplasma contamination in cell lines.
J Immunol Methods 164:91–100

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

15

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C.C. Uphoff and H.G. Drexler

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

2

Eradication of Mycoplasma Contaminations

17

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).

18

C.C. Uphoff and H.G. Drexler

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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Eradication of Mycoplasma Contaminations

19

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.

20

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

2

Eradication of Mycoplasma Contaminations

Mycoplasma-infected Cell Line

21

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.

22

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.

2

Eradication of Mycoplasma Contaminations

23

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).

24

C.C. Uphoff and H.G. Drexler

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

2

Eradication of Mycoplasma Contaminations

25

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).

26

C.C. Uphoff and H.G. Drexler

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

Chapter 3
STR DNA Typing of Human Cell Lines: Detection of Intraand Interspecies Cross-Contamination
Wilhelm G. Dirks and Hans G. Drexler
Abstract
Inter- and intraspecies cross-contaminations (CCs) of human and animal cells represent a chronic problem
in cell cultures leading to false data. Microsatellite loci in the human genome harboring short tandem
repeat (STR) DNA markers allow individualization of cell lines at the DNA level. Thus, fluorescence polymerase chain reaction amplification of STR loci D5S818, D13S317, D7S820, D16S539, vWA, TH01,
TPOX, CSF1PO, and Amelogenin for gender determination is the gold standard for authentication of
human cell lines and represents an international reference technique. The major cell banks of the USA,
Germany, and Japan (ATCC, DSMZ, JCRB, and RIKEN, respectively) have built compatible STR databases to ensure the availability of STR reference profiles. Upon determination of an STR profile of a
human cell line, the suspected identity can be proven by online verification of customer-made STR data
sets on the homepage of the DSMZ institute. Furthermore, an additional tetraplex PCR has been established to detect mitochondrial DNA sequences of rodent cells within a human cell culture population.
Since authentic cell lines are the main prerequisite for rational research and biotechnology, the next sections describe a rapid and reliable method available to students, technicians, and scientists for certifying
identity and purity of human cell lines of interest.
Key words: Authentication, Cross-contamination, DNA STR typing, Human cell lines, mtDNA typing, Misidentification, Quality control

1. Introduction
Most facilities culturing cells use multiple cell lines simultaneously.
Because of the complexity of experimental designs today and the
increasing use of cell lines in science and biotechnology, the possibility of inadvertent mixing of cell lines during the course of dayto-day cell culture is always present. Cross-contaminations (CCs)

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_3, © Springer Science+Business Media, LLC 2013

27

28

W.G. Dirks and H.G. Drexler

may be intraspecies, when two genetically different cell lines of
human origin are cultured in one population, or interspecies, when
animal cells invade human cell cultures. Interspecies and intraspecies CCs represent a widespread problem and alerts on misidentified
or cross-contaminated cell lines are most often simply ignored by
the scientific community (1).
Several factors contribute to the increasing problem of crosscontaminated cell cultures worldwide. The high incidence (~15%)
of CC among cell lines obtained directly from original investigators or from secondary sources implies that the majority of false cell
lines arise in the originators’ own laboratories (2). In addition to
failure to correctly identify cells during the establishment of new
cell lines, neglect of quality control procedures and disregard for
good cell culture practices are the key causes of CC in new cell
lines. In addition, less attendance to rules at the bench side is
another driving force behind the widespread CC since many
supervisors are not meeting their obligations and allow Ph.D. students and technicians who are only briefly and poorly introduced
to adequate cell culture techniques to handle multiple cell lines.
Furthermore, information on cell lines exchanged between
scientific collaborators is normally simply assumed to be correct. A plethora of reports unmasking bogus cancer cell lines,
including members of the NCI-60 panel used to generate reference baseline transcriptional drug responses, has triggered calls for
remedial action (3, 4).
While intraspecies CCs of human cells have been widely
reported (5, 6), the problem of contaminating animal cells within
a human culture has been neglected. The scientific authentication
service of DSMZ demonstrates that both types of CCs are pervasive. Evaluation of the identities of over 500 cell lines from external
customers shows that the incidence of intraspecies CC is ~10%
while the incidence of interspecies CC is ~6%. These results highlight the need for a standard authentication procedure for cell line
identity and purity testing.
Researchers using transgenic animal technology utilize various rodent cell lines. The most relevant cell lines for the biotechnology industry, BHK-21 and CHO, were derived from Syrian
and Chinese hamsters, respectively. To detect contamination of
human cell cultures by a broad spectrum of rodent lines, we
developed a PCR-based method for detecting mitochondrial
DNA sequences (mtDNA) from mouse, rat, and Syrian and
Chinese hamster cells. A test to both generate an STR profile of
human cells and detect animal cells represents a major and novel
advance in detecting CCs and thereby decreasing the use of false
cell lines.

3

STR DNA Typing of Human Cell Lines…

29

2. Materials
All solutions should be prepared in water that has a resistivity of
18.2 MΩ-cm and total organic content of less than five parts per
billion. This standard is referred to as “distilled water” in this text.
2.1. Preparation
of High-MolecularWeight DNA

1. Phosphate-buffered saline (PBS): 140 mM NaCl, 27 mM KCl,
7.2 mM Na2HPO4 × 12 H2O, 14.7 mM KH2PO4, pH 7.2,
autoclaved.
2. Absolute isopropanol.
3. Absolute ethanol.
4. TE 10/1: 10 mM Tris, 1 mM EDTA, pH 8.0, prewarmed to
50°C.
5. High Pure PCR template preparation kit (Roche).
6. Water bath prewarmed to 72°C.
7. Standard tabletop microcentrifuge capable of 13,000 × g centrifugal force.
8. Standard spectral photometer for determination of DNA
concentration.

2.2. Multiplex STR
Typing

Fluorescent labeling of the PCR primers permits the multiplexing
of STR loci, even when alleles fall into the same size range, by
labeling the overlapping loci with differently colored fluorescent
dyes. Group I primers are labeled with one specific dye, which
should be different from the dye used for group II primers or for
the size standard. The choice of specific dyes depends on the capillary electrophoresis unit available. See Tables 1 and 2 for a list of
primers. Only one primer of a pair of an STR locus should be
labeled at the 5¢-end, regardless if it is the forward or reverse primer
(see Note 1).
1. Thermal cycler (any supplier).
2. Standard tabletop microcentrifuge or centrifuge capable of
spinning 96-well plates.
3. 0.2 mL reaction tubes.
4. Fluorescently labeled primer pairs for human DNA typing (see
Table 1 for a list of primer sequences) or for animal mtDNA
detection (see Table 2 for a list of primer sequences) concentrated at 100 mM in TE (10/1) as a stock solution and stored
at −20°C.
5. Working solutions of labeled primers, aliquoted at 10 mM in
small amounts (~ 25–50 mL aliquots) and stored frozen at
−20°C (see Note 2).

Table 1
STR primer sequences for human DNA typing
STR primer group I (dye I) for human DNA typing
STR

Locus

Sequence

D16S539

16q22-24

Forward: 5¢-GGG GGT CTA AGA GCT TGT AAA AAG;
Reverse: 5¢-GTT TGT GTG TGC ATC TGT AAG CAT GTA TC

D13S317

13q22-q31

Forward: 5¢-ACA GAA GTC TGG GAT GTG GAG GA;
Reverse: 5¢-GGC AGC CCA AAA AGA CAG A

D5S818

5q21-q31

Forward: 5¢-GGT GAT TTT CCT CTT TGG TAT CC;
Reverse: 5¢-AGC CAC AGT TTA CAA CAT TTG TAT CT

D7S820

7q11.21-22

Forward: 5¢-ATG TTG GTC AGG CTG ACT ATG;
Reverse: 5¢-GAT TCC ACA TTT ATC CTC ATT GAC

STR primer group II (dye II) for human DNA typing
STR

Locus

Sequence

CSF1

5q33.3-34

Forward: 5¢-AAC CTG AGT CTG CCA AGG ACT AGC;
Reverse: 5¢-TTC CAC ACA CCA CTG GCC ATC TTC

TPOX

2p23-2pter

Forward: 5¢-ACT GGC ACA GAA CAG GCA CTT AGG;
Reverse: 5¢-GGA GGA ACT GGG AAC CAC ACA GGT TA

TH01

11p15-15.5

Forward: 5¢-ATT CAA AGG GTA TCT GGG CTC TGG;
Reverse: 5¢-GTG GGC TGA AAA GCT CCC GAT TAT

vWA

12p12-pter

Forward: 5¢-CTA GTG GAT GAT AAG AAT AAT CAG TAT GTG;
Reverse: 5¢-GGA CAG ATG ATA AAT ACA TAG GAT GGA TGG

Amel

Xp22.1-22.3
Yp11.2

Forward: 5¢-ACC TCA TCC TGGG CAC CCT GGT T;
Reverse: 5¢-AGG CTT GAG GCC AAC CAT CAG

Alternate primer sequences for amplification of human STRs, as well as information on the PCR products and allele
sizes, are available at http://www.cstl.nist.gov/div831/strbase

Table 2
STR primer sequences for animal mtDNA detection
STR primer group III (dye III) for animal mtDNA detection
Species

PCR fragment

Primer sequences

Mouse

300 bp mtDNA

Forward: 5¢-AGG ATT CCC AAT CGT CGT AGC
Reverse: 5¢-TGT AAT TAC GGC TCC AGC TCA

Rat

500 bp mtDNA

Forward: 5¢-CAA TCC ACC AAG CAC AAG TG
Reverse: 5¢-CCCCAACCGAAATTTGGTAGTTC

C. Hamster

605 bp mtDNA

Forward: 5¢-CCG GCG TAA AAC GTG TTA TAG ACT
Reverse: 5¢-GTA TTA GGT ATA ATA TCG GCA GTC

S. Hamster

245 bp mtDNA

Forward: 5¢-GAC CTC TTA GGT GTA TTC CTA C
Reverse: 5¢-GTA TTA GGT ATA ATA TCG GCA GTC

Information on the PCR products and allele sizes is available at http://www.cstl.nist.gov/div831/strbase

3
2.2.1. Hot Start Nonaplex
PCR for Human DNA Typing

STR DNA Typing of Human Cell Lines…

31

In addition to the equipment and reagents listed in Subheading 2.2,
the following reagents are required:
1. Master mix consisting of 25 mL per reaction for each sample,
plus one additional reaction for every ten samples. For a single
reaction, the components are as follows:
(a) 10 pmol of STR primer for human DNA typing (1 mL of
a 10 mM working solution).
(b) 2.5 mL 10× Hot start PCR buffer (any supplier).
(c) 1 mL dNTP (5 mM stock solution).
(d) 0.2 mL (1 unit) hot start Taq polymerase (any supplier).
(e) 19.5 mL distilled water.
2. Positive control DNA, e.g., HeLa DNA.
3. Negative control: Distilled water.
4. Test DNA.

2.2.2. Hot Start Tetraplex
PCR for Animal mtDNA
Detection

In addition to the equipment and reagents listed in Subheading 2.2,
the following reagents are required:
1. Master mix containing 25 mL per reaction for each sample,
plus one additional reaction for every ten samples. For a single
reaction, the components are as follows:
(a) 10 pmol of mtDNA primer for animal mtDNA typing
(1 mL of a 10 mM working solution).
(b) 2.5 mL 10× Hot start PCR buffer (any supplier).
(c) 1 mL dNTP (5 mM stock solution).
(d) 0.2 mL (1 unit) hot start Taq polymerase (any supplier).
(e) 19.5 mL distilled water.
2. Positive control DNA, e.g., rodent DNA mixed with HeLa
DNA template.
3. Negative control DNA, e.g., H2O mixed with HeLa DNA
template.
4. Test DNA.

2.3. Capillary
Electrophoresis
for DNA Fragment
Detection

1. Capillary electrophoresis unit (any supplier).
2. Internal size standard kit 400 (Beckman-Coulter).
3. Microtiter plates.
4. Sample loading solution; 0.3 mM EDTA in deionized
formamide.
5. Test samples (PCR amplification products).

32

W.G. Dirks and H.G. Drexler

3. Methods
Multiplex PCR targets multiple locations throughout the genome
and is an ideal technique for DNA typing because the probability
of identical alleles in two individuals decreases with an increase in
the number of polymorphic loci examined (7). The ability to
fluorescently label PCR primers permits the multiplexing of STR
loci which may have alleles that fall into the same size range, by
labeling the overlapping loci with differently colored fluorescent
dyes that can then be resolved spectrally.
General rules to avoid DNA carryover contamination should be
strictly followed. DNA extraction should be carried out using
equipment (pipettes, microcentrifuge, etc.) which is independent
from the PCR setup. Optimally, this laboratory is separated from
those rooms where the PCR reaction is set up or the PCR products
are analyzed. All reagents should be stored in small aliquots to
provide a constant source of uncontaminated reagents. New aliquot batches should be tested and compared for quality prior to
any use. Re-amplifications should never be carried out. If possible,
PCR reactions should be set up in a designated working station or
a hood capable of irradiating used pipettes, tips, and tubes by UV
light. It is highly recommended that gloves are worn during the
whole procedure.
3.1. Preparation
of High-MolecularWeight DNA

Briefly, cells are lysed during a short incubation time with proteinase K in the presence of a chaotropic salt (guanidiniumhydrochloride), which immediately inactivates all nucleases. Nucleic
acids bind selectively to glass fibers prepacked in the filter tube.
Bound genomic DNA is purified in a series of rapid washing and
spinning steps to remove inhibiting cellular components. Finally,
low-salt elution releases the DNA from the glass fiber cushion.
1. Centrifuge cell culture suspensions containing 3–5 × 106 diploid cells in an Eppendorf tube at 1,000 × g for 4 min in a
14 mL tube (see Notes 3 and 4). Remove the supernatant with
a disposable pipette and discard. Carefully suspend the remaining pellet in 5 mL PBS using a pipette. Repeat centrifugation.
2. Suspend the cell pellet in 200 mL PBS by vortexing. Make sure
that even tiny clumps of cells are carefully re-suspended.
3. Pre-warm the water bath to 72°C.
4. Using the commercially available DNA extraction kit from
Roche, add 200 mL of well-mixed solution I (guanidiniumhydrochloride) to the sample solution and mix by careful
pipetting.
5. Immediately add 40 mL proteinase K, mix well using a vortex,
and incubate at 72°C for at least 10 min.

3

STR DNA Typing of Human Cell Lines…

33

6. Add 100 mL of isopropanol to the sample, mix well, and apply
the whole mixture to a filter tube. Centrifuge for 1 min at
6,000 × g.
7. Discard the flow through, add 500 mL of inhibitor removal
buffer, and centrifuge again for 1 min at 6,000 × g.
8. Discard the flow through, add 500 mL of wash buffer, and
centrifuge again for 1 min at 6,000 × g.
9. Repeat step 8.
10. Add 200 mL of elution buffer preheated to 72°C and centrifuge for 1 min at 6,000 × g. For maximum yield the elution
step should be repeated using 100 mL elution buffer. Resuspend
pellet in TE 10/1 (10 mM Tris, 1 mM EDTA, pH 8.0).
11. Using a standard spectral photometer, adjust the concentration of
genomic DNA to 10 ng/mL per sample for a diploid cell line.
12. Store genomic DNA at 4°C (see Note 5).
3.2. Hot Start Nonaplex
PCR of Genomic DNA
and Tetraplex PCR of
Mitochondrial DNA

In the first step, a multiplexed STR PCR amplification of eight
prominent and highly polymorphic STR loci and one additional
locus for gender determination will be carried out. The STRs of the
loci D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX,
and CSF1PO consist of exclusive tetrameric repeats, which are inherited in a Mendelian way. The combination of 8 STRs increases the
exclusion rate sufficiently to allow the discrimination of one human
cell line from another at the level of 108. Amelogenin (AMEL) is the
most suitable gene for gender determination of samples of human
origin (8). Using specific primers in PCR applications, the sequence
of the X-chromosomal version (AMELX, Xp22.1-Xp22.3) yields a
209 bp amplicon, while the Y-chromosomal gene (AMELY, Yp11.2)
yields a 215 bp DNA fragment, which are easily separated by different electrophoretical techniques (9). Hence, samples from male
sources will show two bands (209/215 bp), while female-derived
cell lines will show only one band (209 bp, see Fig. 1). In a second
step, an independent tetraplex PCR for detection of mtDNA
sequences derived from rodent cell lines is carried out.

3.2.1. Hot Start Nonaplex
PCR for Human DNA Typing

The multiplex PCR described here identifies eight different STR
loci and, in addition, determines gender. The amplification procedure and the parameters are optimized for 0.2 mL reaction tubes
in an i-Cycler thermal cycler (Bio-Rad). It is essential to incorporate the appropriate positive and negative controls (e.g., HeLa
DNA template and H2O, respectively).
When using commercial multiplex STR kits, the specific manuals of Promega Corporation and Applied Biosystems should be
strictly followed. If not using commercial kits, it is important to
optimize the general amplification parameters. A “hot start” PCR
should be always performed in order to activate the Taq DNA

34

W.G. Dirks and H.G. Drexler

Fig. 1. Electropherogram of an interspecies CC of cervical carcinoma line HELA. CC of HELA by mouse, rat, and hamster
cells. 10 ng of genomic DNA from the cell line HELA (DSMZ ACC 057) was analyzed using the nonaplex PCR for human STR
fragment amplification (green and black peaks ) and the tetraplex mtDNA amplification of the rodent lines of mouse, rat,
and Chinese/Syrian hamster as indicated.

Polymerase and to prevent the formation of nonspecific amplification
products. The number of cycles depends on the amount of DNA,
so with the protocol as outlined 30 cycles are recommended for all
samples.
1. Prepare a master mix (see Subheading 2.2.1 for components)
containing 25 mL per reaction for each sample, plus one additional reaction for every ten samples. Include enough for both
a positive and a negative control (water only).
2. Aliquot 25 mL of the master mix into reaction tubes or 96-well
reaction plates, one for each sample.
3. Adjust genomic DNA to a concentration between 0.2 and
1 ng/mL.
4. Add 1 mL of genomic DNA to each tube or well.
5. Centrifuge the tubes/plates for 4 min at 600 × g.
6. Program the thermal cycler as follows:
Cycle 1 (×1):

94°C for 3 min

Cycle 2 (×10):

94°C for 30 s, 60°C for 15 s, 70°C for 45 s

Cycle 3 (×20):

90°C for 30 s, 60°C for 15 s, 70°C for 45 s

Cycle 4 (×1):

60°C for 30 min

Cycle 5:

4°C end

3
3.2.2. Hot Start Tetraplex
PCR for Animal mtDNA
Detection

STR DNA Typing of Human Cell Lines…

35

Detection of animal sequences is carried out using a separate
tetraplex PCR reaction using specific primers for mtDNA sequences
of mouse, rat, and Syrian and Chinese hamster. The main advantage of using mitochondrial versus genomic DNA sequences is the
presence of high amounts of mitochondrial genomes (up to 104 in
liver) compared to a diploid nuclear genome. While equimolar
ratios of DNA sequences allow a detection limit of ~10%, the presence of a single rodent cell can be detected in 1 out of 104 to 105
human cells. It is essential to incorporate the appropriate positive
and negative controls (e.g., each rodent DNA mixed with HeLa
DNA template and H2O mixed with HeLa DNA template,
respectively).
1. Prepare a master mix (see Subheading 2.2.2 for components)
containing 25 mL per reaction for each sample, plus one additional reaction for every ten samples. Include enough for both
a positive and a negative control.
2. Aliquot 25 mL of the master mix into reaction tubes or 96-well
reaction plates, one for each sample.
3. Adjust genomic DNA to a concentration between 0.2 and
1 ng/mL.
4. Add 1 mL of genomic DNA to each tube or well.
5. Centrifuge the tubes/plates for 4 min at 600 × g.
6. Program the thermal cycler as follows:

3.3. Capillary
Electrophoresis
for DNA Fragment
Detection and Allelic
STR Lists

Cycle 1 (×1):

94°C for 3 min

Cycle 2 (×35):

94°C for 4 s, 50°C for 30 s, 72°C for 60 s

Cycle 3 (×1):

72°C for 7 min

Cycle 4:

4°C end

Since DNA possesses a constant mass-to-charge ratio, some form
of separation matrix is needed to separate different sizes of DNA
fragments by their molecular weight. In traditional gel electrophoresis, the requirement for a sieving matrix is met with polyacrylamide or agarose gels. The movement of larger DNA fragments is
impeded relative to that of the smaller DNA fragments as the
molecules migrate through the gel under the influence of an electric field. Polyacrylamide gels are no longer the only slab gel
systems available for resolving STR alleles. A recent publication
demonstrated that small agarose gels have sufficient resolving
power to type tetranucleotide repeats. Even dinucleotide repeats
could be resolved with MetaPhor agarose and detected with SYBR
Green staining (9).
Various automated fluorescence detection systems have been
used for separation, detection, and typing of STR alleles. Full auto-

36

W.G. Dirks and H.G. Drexler

mation of the electrophoresis process with no need to pour the gel
or manually pipet the samples onto the gel is one of the big advantages of capillary electrophoresis (CE). With the higher sensitivity
of laser-induced fluorescence, sample preparation is no longer necessary. Samples are diluted in water or formamide and can be easily
detected. Separation of STR alleles may be performed in a matter
of minutes rather than hours.
1. Aliquot 1 mL of each PCR amplification product into a well in
a microtiter plate.
2. Add 0.25 mL of the internal size standard.
3. Add 29 mL of sample loading solution for a total volume of
30 mL.
4. Run the Capillary Electrophoresis as per the manufacturer’s
instructions.
Using Capillary Electrophoresis, samples are automatically loaded
and analyzed using fragment analysis parameters, which are provided by the supplier of the Capillary Electrophoresis unit (Applied
Biosystems, Beckman-Coulter). In general, fragment analysis
software of any supplier enables the precise determination of
detected alleles resulting in an allelic genotype list. Allelic numbers of STR DNA fragments may be precisely determined by
other techniques or by allelic ladders of the ABI system.
5. Once the sizes are known, determine the respective allele numbers from Table 3 (see Note 6).
3.4. Online Evaluation
of Suspicious STR
Profiles

The main Biological Resource Centers (BRCs) ATCC, DSMZ,
JCRB, and RIKEN have generated large databases of STR cell line
profiles for identity control. In cooperation with the Japanese
BRCs, DSMZ has piloted the generation of the most comprehensive international reference database which is linked to a simple
search engine for interrogating STR cell line profiles (10). A simple
search engine for interrogating STR cell line profiles has been
made available on the Website of DSMZ. Once the problem of
false negatives due to discrepant representation of single STR
alleles—e.g., by losses of heterozygosity and bottlenecking
selection—has been tackled and unambiguous search results are
produced, human cell lines should be consistent with consensus
STR reference data sets. STR profiles of all human cell lines distributed by DSMZ, JCRB, and RIKEN and one-third of the cell lines
distributed by ATCC are now publicly accessible at http://www.
dsmz.de/services/services-human-and-animal-cell-lines/online-stranalysis.html using an interactive database where match-criteria
have been arbitrarily set to 60%. Registered users simply login at
the online-STR-analysis-site on the DSMZ homepage and are
guided through the procedure. Aided by simple prompts, users can
input their own cell line STR data to retrieve best matches with

3

STR DNA Typing of Human Cell Lines…

37

Table 3
Allele organization and sizes of amplified human STR loci
Allele

D5S818 D13S317 D7S820 D16S539

vWA

TH01

3

169

4

173

5

TPOX

CSF1PO

Amelogenin

209 = X

164

212

266

177

220

287

6

114

168

216

270

181

224

291

7

118

172

220

274

185

228

295

8

122

176

224

278

189

232

299

9

126

180

228

282

193

236

303

215 = Y

196

9.3
10

130

184

232

286

118

197

240

307

11

134

188

236

290

122

201

244

311

12

138

192

240

294

126

205

248

315

13

142

196

244

298

130

252

319

14

146

200

248

302

134

256

323

15

150

204

252

306

138

327

16

154

142

331

17

158

146

18

150

19

154

20

158

21

162

22

166

23

170

Nucleotide range and the number of known alleles of each STR loci are summarized. Further information is available at
http://www.cstl.nist.gov/div831/strbase. Regular fragment sizes in base pairs of alleles are printed in bold, variant
alleles are printed in italics

authenticated cell lines listed on the database. Inevitably, reference
profiles remain subject to revision until all commonly held cell lines
have been STR typed across participating repositories. At present,
about 2,342 such cell lines have been STR typed and are represented as reference sets on the database. Armed with this tool,
online verification of cell line identity should prove a vital weapon
to combat the havoc of cell line cross-contamination which has
dogged cancer research since inception.

38

W.G. Dirks and H.G. Drexler

4. Notes
1. If an individual constellation of genomic loci is of interest, the
dye-labeled primer pairs should be tested in single reactions
and the primer mixture adjusted for the generation of similar
peak heights of the measured loci.
2. Working solutions of primers are stable for 1 month at 4°C and
should be kept in lightproof reaction tubes.
3. Prior to isolation of genomic DNA, cell viability of samples
should be analyzed using trypan blue exclusion assay. With the
exception of factor-dependent hematopoietic cell lines, viability
of a cultured cell line should not be below 85% at sampling.
4. To avoid retention of PCR inhibitors in biological samples, it
is very important to carefully and completely suspend the cell
pellet in PBS prior to DNA isolation.
5. Quality control of isolated genomic DNA should be carried
out