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CGMH
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Detection and Treatment of Mycoplasma Contamination
in Cultured Cells |
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Hsuan Jung
Shih-Yee Wang1
I-Wen Yang, MS
Ding-Wei Hsueh, MS
Wei-Ju Yang, MS
Tzu-Hao Wang2,3, MD, PhD
Hsin-Shih Wang4, MD, PhD
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Background:
Mycoplasmas, the smallest and simplest prokaryotes that reside
in endosomes of mammalian cells, are widespread contaminants
found in cell cultures. About 30% of all cell cultures, varying
from 15 to 80%, are reportedly contaminated with mycoplasmas.
Here, we present our experience in successfully detecting
and treating mycoplasmal infection in various cell lines.
Methods:
The nested polymerase chain reaction (PCR) detection and microscopic
examination, including phase-contrast, fluorescent, as well
as differential interference contrast, were used for detecting
potential mycoplasma contamination of cell lines used in our
laboratory. As soon as mycoplasma was identified, antibiotic
treatment was initiated.
Results:
Mycoplasmal contamination was detected in six of 15 cell lines
using the nested PCR amplification of mycoplasma DNA, which
was further demonstrated using 4, 6-Diamidino-2-phenylindole
(DAPI) staining and fluorescent microscopy. Alternate treatment
with two antibiotics, macrolide (tiamulin) and tetracycline
(minocycline), effectively eliminated mycoplasma, which was
validated by both PCR and microscopic studies.
Conclusions:
The nested PCR using genomic DNA extracted from cultured cells
as templates is a rapid and sensitive method for detecting
mycoplasma contamination. Treatment with combined antibiotics
can completely eradicate mycoplasmal infection from cultured
cells. For the ease of use, PCR and/or DAPI staining appear
suitable for detecting potential mycoplasmal contamination
in laboratories that rely heavily on the cell culture system.
(Chang Gung Med J 2003;26:250-8)
Key words:
cell culture, mycoplasmal contamination.
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Mycoplasmas, the smallest (0.3-0.8 µm diameter)(1) and simplest
prokaryotes that reside in endosomes of mammalian cells, are
widespread contaminants found in cell cultures. About 30% of
all cell cultures, varying from 15 to 80%, are reportedly contaminated
with mycoplasmas.(2) The concentration can be as high as 107
colony-forming units (CFU) per milliliter.(3) Unlike other commonly
prevalent bacteria, mycoplasmas are bound by a triple-layered
membrane and lack of rigid cell walls, making them resistant
to penicillin and its analogues.
Mycoplasmal infection may deprive host cells of arginine,(4)
an essential amino acid which mycoplasmas use as an energy source,
and alter incorporation of nucleic acid precursors into the
host cells.(5,6) Attachment of mycoplasmas may cause host cells
to leak and interfere with membrane-receptor function as well
as signal transduction.(7) It may impose a deleterious effect
on transient transfection which involves endocytosis of plasmid
DNA.(8)
Mycoplasma-infected cells frequently show stunted, abnormal
growth and 'moth-eaten' edges of the monolayer. Other cells
may show cellular changes similar to nutrient deprivation which
can be reversed by frequently changing the medium.(1) Unfortunately,
not all contamination can be detected by examining the changes
of morphology.
Many detection methods have been developed, including microbiological
cultivation on broth and agar, fluorescence DNA staining using
4', 6-diamidino-2-phenylindole (DAPI) or Hoechest dyes,(9,10)
nucleic acid hybridization with a probe specific for mycoplasmal
rRNA,(11) enzyme-linked immunosorbent assay (ELISA) with mycoplasma-specific
polyclonal antibodies,(12) immunofluorescence staining,(13)
biochemical detection utilizing 6-methyl purine deoxyriboside
(6-MPDR, an analogue of adenosine, which can be converted by
mycoplasma to 6-methyl purine and 6-methyl purine riboside,
both toxic to mammalian cells),(14) and polymerase chain reaction
(PCR).(15,16) Among them, PCR is considered to be rapid and
sensitive. Hopert et al. reported that PCR produced significantly
fewer false-negative or false-positive results than DAPI DNA
fluorescence staining, immunostaining with a monoclonal antibody,
ELISA, and DNA-RNA hybridization in solution. Moreover, PCR
also showed better sensitivity when samples were diluted.(17)
Comparisons among the four detection methods indicated that:
(a) the microbiological culture is time-consuming, taking 1
to 4 weeks; (b) DNA fluorescent staining can be difficult to
interpret due to the presence of contaminating bacteria or broken
nuclei; (c) nonspecific signal interference may be caused by
the presence of cross-reacting gram-positive bacteria in hybridization
process; and (d) PCR can rapidly detect as few as 3-100 CFU/ml
with good specificity.(18) Among different species of mycoplasma,
PCR detected 20-180 CFU/ml reliably.(16) Most PCR methods employ
primers that hybridize with 16S or 23S rRNA conserved to prokaryotes,
and amplify the spacer region between 16S and 23S rRNA. The
length and sequence of the spacer region, which differs from
species to species, can be used to identify the contaminants
by running an agarose gel or performing a second PCR.
Once contamination is detected, completely autoclaving the incubator
and replacing with mycoplasma-free stock are recommended. However,
when another mycoplasma-free stock is not available, eradicating
mycoplasma from the infected cells is necessary. Mycoplasma
eliminating methods can be classified into four types: (a) physical
procedures, such as heat treatment or photo sensitizing; (b)
chemical procedures, such as washings with ether-chloroform
or culturing in medium containing 6-MPDR; (c) immunological
procedures, such as treatment with specific anti-mycoplasma
antisera or exposure to complement; and (d) chemotherapeutic
procedures, such as antibiotic treatment in standard culture
or soft agar cultivation.(19) The addition of antibiotics into
the medium seems to be attractively simple and inexpensive,
with the efficacy reportedly to be more than 75%.(20) Several
anti-mycoplasmal agents, primarily quinolone and tetracycline,
are commercially available. These reagents inhibit either nucleic
acid synthesis or protein synthesis.(19) In the experiment by
Fleckenstein,(21) all of the mycoplasma-positive cultures resistant
to a first antibiotic could be cleaned up using a second treatment
with a different antibiotic. From the comparison of several
antibiotic regimens,(19-22) BM-cyclin (trade name), which is
a combination of tiamulin and minocycline that both inhibiting
protein synthesis, has shown to have better treatment efficiency
than Sparflox, Enroflox, Ciprololx (a DNA gyrase inhibitor),
or the mycoplasma removing agent that is a DNA gyrase inhibitor.
For years at our laboratory, we have been using more than a
dozen cell lines for studying reproductive physiology and tumor
biology. However, recent observations on the slow growth of
the cells, quick acidification of the media, and poor efficiency
of the transfection cautioned us about the potential contamination
of mycoplasma in the cells. Here, we present our experience
in successfully detecting and treating mycoplasmal infection
of various cell lines.
METHODS
Cell cultures
Cell lines that were screened in this study included human
ovarian cancer cells (BG1, SKOV3, OVCAR3, BR, 67R, CaOV3),
choriocarcinoma (JEG3, BeWo), breast cancer cells (MCF-7,
T47D), endometrial cancer cells (KLE, RL-952), cervical cancer
cells (HeLa), embryonal kidney cells (293), and Chinese hamster
ovary cells (CHO). After being thawed, one part of the cell
line stock was directly centrifuged then cultured, another
part underwent DNA extraction. For cell cultures, cell lines
(Table 1, all adherent cell lines), were cultured in 25 cm2
culture flasks containing D-MEM/F-12 (Gibco BRL Life Technologies,
#12400-024, Karlsruhe, Germany) medium with 10% fetal bovine
serum (FBS; Gibco BRL, #16000-044), 100 units/ml of penicillin
and 100 µg/ml of streptomycin (Gibco BRL, #15140-122). All
cultures were maintained at 37oC in a humidified incubator
containing air and 5% CO2.
DNA extraction
DNA of each cell line was extracted using QIAamp DNA Blood
Midi Kit (Qiagen, #51183, Hilden, Germany) according to the
manufacturer's instruction.
Nested Polymerase chain reaction (PCR)
Mycoplasmal contamination was detected using a PCR Mycoplasma
Detection Set (Takara, #6601, Shiga, Japan). Briefly, the
first PCR amplified the spacer region between the 16S and
23S mycoplasmal rRNA- the forward primer (5'-ACACCATGGGAGCTGGTAAT-3')
and reverse primer (5'-CTTCATCGACTTTCAGA-CCCAAGGCAT-3'). The
second run of nested PCR was then performed using a forward
primer (5'-GTTCTTTGAAAACTGAAT-3') hybridizing with a conservative
sequence on the spacer region and a reverse primer (5'-GCATCCACCAAAAACTCT-3')
hybridizing with 23S rRNA. Depending on the different mycoplasma
species, the product lengths of the first PCR ranged from
369 to 681 bp, and from 145 to 237 bp for the second PCR.
For each cell line, 200-900 ng of extracted DNA or 10 µg/ml
of medium collected from the cells that had been cultured
for at least 3 days were used as PCR templates. Negative controls
were included in every PCR experiment to ensure the absence
of contamination. Taq polymerase from Takara (Takara, #R001A),
and a thermocycler (Applied Biosystems GeneAmp PCR System
9700; Perkin Elmer Applied Biosystems, Weiterstadt, Germany)
were used. PCR products were analyzed using agarose gel electrophoresis
(1.5% and 2% for first and second runs of PCR, respectively)
and ethidium bromide staining.
4,6-Diamidino-2-phenylindole (DAPI) staining
The fluorescent DAPI dye (Sigma, #D-8417, Deisenhofen, Germany)
was dissolved in water to make the 1 mg/ml stock. The working
solution was freshly prepared by diluting the stock DAPI into
1 µg/ml with methanol. Cells cultured on chamber slides (Nunc,
#154461, Wiesbaden, Germany) coated with fibronectin were
rinsed once with the working solution, incubated with the
working solution in 37oC for 15 minutes, then rinsed once
with methanol. Slides were mounted with glycerol and examined
under a fluorescence microscope with 340/380 nm excitation
filter and LP 430 nm barrier filter (Olympus, BX50, Japan).
Alternatively, a confocal microscope (Leica TCS SP2, Germany)
was used to capture the isolated and overlapped images of
differential interference contrast (DIC) and fluorescence.
Mycoplasma elimination
Two mycoplasma infected cell lines, BR and 67R, were treated
with BM-cyclin (Roche, #799050, Mannheim, Germany) according
to the supplier's recommendations. Briefly, BM-cyclin I (macrolide
tiamulin) was added to a final concentration of 10 µg/ml for
3 days followed by BM-cyclin II (tetracycline minocycline)
at 5 µg/ml for 4 days. The treatment cycle was repeated three
times.
RESULTS
Detection of mycoplasmal contamination with PCR
Among the 15 cell lines analyzed (Table 1), six were detected
as mycoplasma-positive, as shown by the amplified fragments
of 488 bp and 211 bp in first and second runs of PCR, respectively
(Fig. 1). These results also indicated that all of the contaminated
cell lines were infected with the same species, M. hyorhinis.
Elimination of mycoplasmas with BM-cyclin treatment
Before treatment, both cell lines showed severely inhibited
growth. When stained with DAPI, mycoplasma DNA scattered in
the cytoplasm was clearly observed. In the severely infected
cells, mycoplasma even leaked out of the host cells (Figs.
2 and 3). The cell lines were examined using both DAPI staining
and PCR before as well as after BM-cyclin treatment (Fig.
4). The DAPI staining demonstrated that treated cells were
completely free of mycoplasma infection (Figs. 2 and 3). Nevertheless,
the PCR results revealed residual amounts of mycoplasma DNA
within the BR cells, as shown in lane 8 in the lower panel
of Figure 4. The residual mycoplasmal DNA apparently belonged
to dead mycoplasma that imposed no harm on the treated cells.
The follow-up PCR using DNA extracted from the treated cells
that were continuously cultured for 3 more weeks did not detected
mycoplasma DNA anymore (data not shown).
DISCUSSION
Although more convenient, direct application of culture medium
for PCR detection, as recommended by the PCR Mycoplasma Detection
set (Takara), was not as sensitive as using extracted DNA
as templates. When culture medium was used as the source of
the PCR templates, no contaminations (67R, HeLa, CHO) were
detected on any of the three cell lines until the second run
of PCR. However, when DNA was used as templates, positive
results were found on the first run (Fig. 1). Similarly, the
culture medium of JEG3 cells showed negative PCR results but
contamination was detected when the DNA was examined. When
DNA was applied, most contamination was detected on the first
run of PCR. However, very slight infections, such as that
of JEG3, may not give a visible band (Fig. 1). Thus, a nested
PCR is still essential for improving the sensitivity even
when DNA is used.
We chose BM-Cyclin for mycoplasma elimination because it was
reported to have better efficacy than other antibiotics. In
our laboratory, three cycles of alternative treatment effectively
eliminated mycoplasma from ovarian cancer 67R and BR cells
(Fig. 4). The same success was achieved for other contaminated
cells too (data not shown).
The general practice of managing mycoplasmal contamination
in cultured cell lines was to discard the contaminated cells
and replace with new ones, unless those cells could not be
obtained elsewhere as for the BR and 67R ovarian cancer lines
in our case. Resources of cell lines include American Type
Culture Collection (ATCC, http://www.atcc.org) internationally
or from Bioresources Collection and Research Center (BCRC,
http://www.ccrc.firdi. org.tw) in Taiwan.
Some undesired side effects of antibiotic treatment may occur
including(19) (a) strong cytotoxicity leading to loss of culture;
(b) growth inhibition under treatment; (c) loss of special
cellular characteristics; and (d) clonal selection of treated
cells. During treatment, we observed that cell death occurred
more frequently in cultures with low cell densities and when
antibiotics were added before cells attached to the culture
flask. Both situations made cells more susceptible to the
cytotoxic effects of BM-Cyclin. Inhibited growth was also
observed but it was manageable and reversible. The inhibition
was minimal when cells were grown at a higher density. In
addition, after three cycles of BM Cyclin treatment were completed,
the cell growth rates resumed. Nevertheless, we still emphasize
the importance of having back-up vials of cells in stock in
case the antibiotic treatment kills the cells.
Concluded from these experiences, we suggest that a nested
PCR using genomic DNA extracted from cultured cells as templates
is a rapid and sensitive method to detect mycoplasma contamination
and the treatment of BM-Cyclin can completely eradicate mycoplasmal
infection from cultured cells. For the ease of use, PCR and/or
DAPI staining appear suitable for detecting potential mycoplasmal
contamination in laboratories that heavily rely on the cell
culture system.
Acknowledgements
The study was supported by grants CTRP1007 (awarded to T.H.
Wang) and CTRP1008 (awarded to H.S. Wang) from the Chang Gung
Memorial Hospital.
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From the Department of Obstetrics and Gynecology, Chang
Gung Memorial Hospital, Taipei; 1Naperville North High School,
Naperville, Illinois, USA; 2Genomic Medicine Research Core
Laboratory, Chang Gung Memorial Hospital; 3School of Traditional
Chinese Medicine, College of Medicine, Chang Gung University;
4Graduate Institute of Clinical Medical Sciences, College
of Medicine, Chang Gung University, Taoyuan.
Received: Oct. 22, 2002
Accepted: Jan. 17, 2003
Address for reprints: Dr. Tzu-Hao Wang, Department of Obstetrics
and Gynecology, Chang Gung Memorial Hospital. 5, Fushing Street,
Gueishan Shiang, Taoyuan, Taiwan 333, R.O.C.
Tel.: 886-3-3281200 ext. 8984
Fax: 886-3-3288252
E-mail: knoxtn@ms34.hinet.net
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