Jpn. J. Infect. Dis., 61, 9-12, 2008
Original Article
Characterization of Mouse 3T3-Swiss Albino Cells Available in Japan:
Necessity of Quality Control When Used as Feeders
Takamasa Takeuchi, Lina Wang1, Seiichiro Mori, Keiichi Nakagawa2,
Hiroshi Yoshikura and Tadahito Kanda*
Center for Pathogen Genomics, National Institute of Infectious Diseases, Tokyo 162-8640;
2
Department of Radiology, Graduate School of Medicine, The University of Tokyo,
Tokyo 113-8655, Japan; and 1Department of Obstetrics and Gynecology, The First Affiliated Hospital,
Jilin University, Changchun, China
(Received October 2, 2007. Accepted October 12, 2007)
SUMMARY: Mouse 3T3-Swiss albino cells are widely used as feeder cells to culture the human epidermis for
the treatment of burns. To minimize the risk of xenoinfection, quality control of the feeder cells is required in
the Japanese guidelines on regenerative medicine using feeder cells. We characterized three lots of 3T3-Swiss
albino cells that are publicly or commercially available in Japan. One lot, which propagated more rapidly than
the other two without showing typical contact inhibition, was found to release endogenous murine leukemia
virus upon iododeoxyuridine-treatment. Southern blotting of restriction fragments showed that the rapidly growing
lot consisted of genetically altered cells that had probably emerged during the passages. The data support the
guidelines that recommend the quality control of each lot of 3T3-Swiss albino cells if they are to be used clinically.
are candidates to be used as feeders for culturing the clinical
human epidermis. In this study we obtained 3T3-Swiss
albino cells from one commercial and two public sources and
examined them for their growth characteristics and their
capacity to produce endogenous MLV. In addition, we confirmed that 3T3-Swiss albino cells were distinguishable from
NIH/3T3 cells or BALB/3T3 cells by Southern blotting of
restriction fragments.
INTRODUCTION
Mouse 3T3-Swiss albino cells, particularly the J2 clone,
are widely used as feeder cells for culturing the human
epidermis, which is clinically used for the treatment of burns
(1-3). Since the epidermis is produced in direct contact with
the feeder layer, it usually contains a small number of the
feeder cells. Thus, it is important to perform quality control
of the feeder cells to minimize the risk of xenoinfection.
The proper method of ensuring quality control in Japan is
described in the “Public Health Guidelines on Infectious
Disease Issues in Skin-graft Produced by Co-cultivation of
Human Keratinocytes with Mouse Feeder Cells” (http://www.
mhlw.go.jp/general/seido/kousei/i-kenkyu/isyoku2/sisin.html
[in Japanese]). The feeder cells should be negative for abnormal growth and for the induction of endogenous murine
leukemia virus (MLV), which is known to be present in the
cellular genome (4). An efficient test for the MLV induction
is to treat cells with iododeoxyuridine (IdU) (5,6).
3T3 cells were originally established from Swiss albino
mouse embryos by Todaro and Green in the 1960s, and now
they are being circulated throughout the world through
several channels (7). Each lot of the cells has been repeatedly
passaged and could be genetically altered. Furthermore, the
situation is complicated by the existence of other cells with
similar names containing 3T3, which is an abbreviation for
the passage scheme of seeding 3×105 cells on a 50-mm dish
every 3 days. Other mouse fibroblast cell lines have been
established through the same “3T3” passage scheme, namely
NIH/3T3 cells (8) and BALB/3T3 cells (9). These cells must
be recognized accurately.
It is important to determine the quality of the 3T3-Swiss
albino cells available in Japan at present, because the cells
MATERIALS AND METHODS
Cells: The frozen stocks of 3T3-Swiss albino (Cat.CCL92), NIH/3T3 (Cat.CRL-1658), and BALB/3T3 clone A31
(Cat.CCL-163) were obtained from the American Type Culture Collection (ATCC) (Manassas, Va., USA). The frozen
stocks of 3T3-Swiss albino (Cat.JCRB9019), NIH/3T3 clone
5611 (Cat.JCRB0615), and BALB/3T3 clone A31 (Cat.
JCRB9005) were obtained from the Japanese Cell Research
Bank (JCRB) through the Health Science Research Resources
Bank (Osaka, Japan). The live cultures of 3T3-Swiss albino
(Cat.08 - 092) and NIH/3T3 (Cat.09 - 1658) were purchased
from the Dainippon Sumitomo Pharma Co., Ltd. (Osaka,
Japan). The frozen stocks of NIH3T3-3-4 (Cat.RCB1862)
and BALB/3T3 clone A31 (Cat.RCB0005) were obtained from
the RIKEN Bioresource Center (RIKEN BRC) (Ibaraki,
Japan). All of the cells were cultured in Dulbecco’s Modified
Eagle Medium containing antibiotics and 10% fetal bovine
serum (growth medium) at 37°C under 5% CO2 in a humidified incubator. For all the experiments, cells were used within
5 passages after reception, and, when necessary, one cycle of
cryopreservation was included.
Growth assay: Cells (1.5 to 2 × 105) were seeded on a 35mm dish (BD, Franklin Lakes, N.J., USA), and 48 h later
they were trypsinized and counted with a Z2 Coulter counter
(Beckman Coulter, Inc., Fullerton, Calif., USA). Similarly,
cells (1 × 105) were seeded on a 60-mm dish (BD) and
counted 72 h later.
Treatment of cells with IdU: Cells (1.5 × 105) in 1.5 ml
*Corresponding author: Mailing address: Center for Pathogen
Genomics, National Institute of Infectious Diseases, 1-23-1
Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel: +81-3-52851111, Fax: +81-3-5285-1166, E-mail: [email protected]
9
growth medium were seeded on a 35-mm dish. The next day,
IdU (30 μg/ml) was added to the medium. Twenty-four h
later the medium containing IdU was removed and the cells
were washed twice with the growth medium. The cells were
cultured in the growth medium, which was harvested daily
and replaced with fresh medium. The harvested medium
was filtered through a 0.45-μm PVDF filter (Milex HV;
Millipore, Billerica, Mass., USA) and kept frozen at –80°C
until a reverse transcriptase (RT) assay was performed.
RT assay: RT activity was measured by using the HS-Mn2+
RT kit (Cavidi Tech AS, Uppsala, Sweden) according to the
manufacturer’s protocol, which contained the following steps:
elongation from the immobilized primer and incorporation
of bromodeoxyuridine (BrdU) by RT, immunodetection of
BrdU, and colorimetric assay. The RT standard provided by
the kit was used to quantitate the RT activity of the samples.
Southern blotting: Cellular DNA was purified by using
QIAGEN genomic tips (QIAGEN GmbH, Hilden, Germany).
After digestion with an appropriate restriction enzyme, DNA
was purified by phenol/chloroform extraction, chloroform
extraction, and ethanol precipitation. Ten microgram of each
sample was electrophoresed on a 1% agarose gel and transferred to a nylon membrane (Hybond XL; GE Healthcare
UK Ltd., Little Chalfont, UK). Radiolabeled probes were
prepared by using Rediprime II (GE Healthcare UK). The
full-length cDNA of MLV (a kind gift from Dr. Yoshihiro
Kitamura) and mouse minisatellite Pc-1 DNA (a kind gift
from Dr. Ryo Kominami) (10) were excised from plasmid
backbone, gel-purified, and used for templates for random
labeling. Hybridization was carried out at 74°C for 1 h in
ExpressHyb (Clontech Laboratories, Inc., Mountain View,
Calif., USA). After hybridization, membranes were washed
in 2 × SSC (1 × SSC contains 15 mM sodium citrate and
150 mM sodium chloride) containing 0.05% sodium dodecyl
sulfate (SDS) at 70°C for 1 h with three changes of the buffer
and then in 0.1 × SSC containing 0.1% SDS at 70°C for 1 h
with one change of the buffer. Phosphoimages were obtained
using Storm860 (GE Healthcare UK).
3T3-J, had lost its contact inhibition and grew to a higher
density (data not shown).
Induction of MLV from 3T3-Swiss albino cells: Endogenous MLV was induced from 3T3-P by stimulation with IdU.
3T3-Swiss albino cells were cultured in the growth medium
containing IdU for 24 h. The culture medium was harvested
and replaced with fresh medium every 24 h, and the RT activity of the harvested medium was measured by a colorimetric
assay (Fig. 2). RT activity was detected only in the IdU-treated
3T3-P culture medium. The peak of RT activity appeared at 1
to 2 days after the IdU-treatment and reached 3 to 4 mU/ml.
Southern blotting of DNA extracted from 3T3-Swiss
albino cells, NIH/3T3 cells, and BALB/3T3 cells: 3T3-Swiss
albino cells, NIH/3T3 cells, and BALB/3T3 cells showed their
distinctive Southern blotting patterns (Fig. 3). The patterns
obtained from EcoRI-digested DNA samples probed with
MLV DNA (Fig. 3A) or Pc-1 DNA (Fig. 3B) were similar in
NIH/3T3 cells and BALB/3T3 cells, but not in 3T3-P cells.
However, those obtained from HaeIII-digested (Fig. 3C),
HinfI-digested (Fig. 3D), and MboI-digested (Fig. 3E) DNA
samples probed with Pc-1 DNA were unique to each cell line.
Among 3T3-Swiss albino cells, 3T3-A and 3T3-J showed
a similar pattern, but there were extra hybridization signals
specific to 3T3-P alone (indicated with arrowheads in Fig. 3,
B to E). 3T3-P is not a cloned cell line, but the majority of the
cells are likely to have the same genotype because the 3T3P-specific signal was as intense as other signals common to
all 3T3-Swiss albino cells. It is very likely that a single cell
with the genetic alteration had emerged and become dominant
over the others during the passages. The genetic alteration is
probably associated with the capacity to produce endogenous
Table 1. Growth of 3T3-Swiss albino cells
Lot of cells
Fold-increase of cell number
Cell growth at 48 h in a 35-mm dish seeded at
1.5 - 2 × 105 cells
3T3-P
6.7 ± 0.2*
3T3-A
2.4 ± 0.1
3T3-J
3.7 ± 0.2
RESULTS
Cell growth at 72 h in a 60-mm dish seeded at
1 × 105 cells
3T3-P
7.2 ± 0.6*
3T3-A
1.4 ± 0.1
3T3-J
3.0 ± 0.6
Morphology and growth of 3T3-Swiss albino cells: During the initial propagation of the cells, we noticed that the
purchased 3T3-Swiss albino cells (designated 3T3-P) were
morphologically different from the other two (designated 3T3A and 3T3-J) (Fig. 1); the 3T3-P cells were round with irregular projections. The 3T3-P cells grew much faster than
the 3T3-A or 3T3-J cells (Table 1). 3T3-P, unlike 3T3-A and
*: P < 0.01.
Average of four measurements with standard
deviation is shown.
Fig. 1. Phase-contrast micrographs of 3T3-Swiss albino cells used in this study. (A) 3T3-Swiss albino cells obtained from ATCC
(designated 3T3-A). (B) 3T3-Swiss albino cells obtained from JCRB (3T3-J). (C) 3T3-Swiss albino cells purchased from
Dainippon Sumitomo Pharma Co., Ltd. (3T3-P). The micrographs were taken with a BZ-8000 microscope (Keyence, Osaka,
Japan). Scale bar represents 50 μm.
10
Fig. 2. RT activity of culture supernatant of 3T3-Swiss albino cells
treated with iododeoxyuridine (IdU). 3T3-Swiss albino cells were
incubated in the medium with (treated) or without (untreated) IdU
(30 μg/ml) for 24 h. “Day 0” denotes culture medium at the end of
IdU-treatment. Shown are the representative data from 4 independent
experiments. The sensitivity of the assay was about 0.08 mU/ml.
MLV after the IdU-treatment (Fig. 2).
All NIH/3T3 cells showed the same pattern, and one lot of
BALB/3T3 cells differed from the others as in the case of
3T3-Swiss albino cells.
DISCUSSION
In this study, we demonstrated that among three publicly
or commercially available lots of 3T3-Swiss albino cells, the
3T3-P cells were phenotypically and genetically different from
the other two. The data indicate that the 3T3-Swiss albino
cells have the potential to be transformed by unintentional
mutations that occur during the passages and that some mutations are associated with the easy induction of endogenous
MLV. Therefore, the quality control of each lot of the feeder
3T3-Swiss albino cells should be conducted according to the
guidelines.
It is likely that performing standardized passages of the
3T3-Swiss albino cells is important for maintaining the
original cellular characteristics. Todaro and Green cultured
several sets of cells that originated from a set of mouse
embryos using different schemes: 3 × 105 cells on a 50-mm
dish every 3 days (3T3), 3 × 105 cells on a 50-mm dish every
6 days (3T6), and 3 × 105 cells on a 50-mm dish every 12
days (3T12) (7). The resulting three cell lines behaved differently. For example, MLV was released only from 3T12 cells
after a long-term culture (11). Also, MLV was induced by the
BrdU-treatment from a clonal cell line derived from 3T6 (12).
We cannot trace the passage history of 3T3-P, but the following sequence of events is plausible. During the passages,
a faster-growing mutant cell emerged, gradually became a
major species, and finally dominated. Changes in the growth
property might be difficult to recognize without setting appropriate controls, because the mutant cells probably outgrow
the other cells gradually, and the loss of contact inhibition is
not detected during routine passages. Therefore, the bank
system, which is useful for minimizing repeated passages of
qualified cells using the master cell-bank and the working
cell-bank, is strongly recommended.
Southern blotting is primarily useful for the identification
Fig. 3. Southern blotting of the restriction fragments. (Panel A) EcoRIdigested DNA samples were hybridized with MLV probe. lane 1,
3T3-Swiss albino from ATCC (3T3-A); lane 2, 3T3-Swiss albino
from JCRB (3T3-J); lane 3, 3T3-Swiss albino from Dainippon
Sumitomo Pharma Co., Ltd. (3T3-P); lane 4, NIH/3T3 from ATCC;
lane 5, NIH/3T3 clone 5611 from JCRB; lane 6, NIH3T3-3-4 from
RIKEN BRC; lane 7, NIH/3T3 from Dainippon Sumitomo Pharma
Co., Ltd.; lane 8, BALB/3T3 clone A31 from ATCC; lane 9, BALB/
3T3 clone A31 from JCRB; lane 10, BALB/3T3 clone A31 from
RIKEN BRC. (Panel B) EcoRI-digested DNA samples were hybridized
with Pc-1 probe. The sample applied to each lane was same with the
Panel A. (Panel C) HaeIII-digested DNA samples were hybridized
with Pc-1 probe. The sample applied to each lane was same with the
Panel A. (Panel D) HinfI-digested DNA samples were hybridized
with Pc-1 probe. The sample applied to each lane was same with the
Panel A. (Panel E) MboI-digested DNA samples were hybridized
with Pc-1 probe. The sample applied to each lane was same with the
Panel A. 3T3-P specific signals are highlighted with white arrowheads
(B-E). The positions of nine larger StyI-digested lambda DNA fragments are marked at the left end of each picture. The sizes of the
fragments (in kbp) are 19.33, 7.74, 6.22, 4.26, 3.47, 2.69, 1.88, 1.49
and 0.93.
of cell strains and for the detection of some, but by no means
all, genetic alterations, as was done in this study. In such cases,
adequate selection of the restriction enzymes and careful
comparison with the reference patterns are important for
detecting the genetic alterations.
11
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39-45.
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17, 299-313.
8. Jainchill, J.L., Aaronson, S.A. and Todaro, G.J. (1969): Murine sarcoma
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cells. J. Virol., 4, 549-553.
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SV40. J. Cell. Physiol., 72, 141-148.
10. Mitani, K., Takahashi, Y. and Kominami, R. (1990): A GGCAGG motif
in minisatellites affecting their germline instability. J. Biol. Chem., 265,
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virus: “spontaneous” release by mouse embryo cells after long-term in
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Science, 174, 157-159.
ACKNOWLEDGMENTS
We thank Drs. Yoshihiro Kitamura and Ryo Kominami for their generous
gift of plasmids.
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