Cell Tissue Bank (2014) 15:373–380
DOI 10.1007/s10561-013-9394-9

ORIGINAL PAPER

A new murine osteoblastic cell line immortalizedwith the SV40 large T antigen
Miki Nakamura-Ota • Ryoji Hamanaka •Hiroyuki Yano • Sawako Adachi •Hideaki Sumiyoshi • Noritaka Matsuo •Hidekatsu Yoshioka

Received: 28 March 2013 / Accepted: 16 August 2013 / Published online: 23 August 2013
Ó Springer Science+Business Media Dordrecht 2013

Abstract The murine preosteoblastic cell line,
MC3T3-E1, is widely used to study bone formationand differentiation in vitro. However, this cell line isunstable in culture. The current study was designed toestablish a stable osteoblastic cell line. A mammalian
expression vector carrying the SV 40 large T antigen
was introduced into a primary culture of cells isolated
from the calvaria of newborn mice. Among isolated
cell lines, the MN16 cell line was selected for further
characterization. The MN16 cell line was cultured for
28 days, and compared with the MC3T3-E1 cell linewith or without induction. The expression of bonerelated genes was examined using the real-time RTPCR technique. Alizarin red and von Kossa staining
were used to detect mineralization of nodules in thecultures. The cell line showed the characteristics of

M. Nakamura-Ota  H. Yano  S. Adachi 
H. Sumiyoshi  N. Matsuo  H. Yoshioka (&)
Department of Matrix Medicine, Faculty of Medicine,
Oita University, 1-1 Hasama-machi, Yufu City,
Oita 879-5593, Japane-mail: hidey@oita-u.ac.jp
M. Nakamura-Ota  R. Hamanaka
Department of Cell Biology, Faculty of Medicine, Oita
University, 1-1 Hasama-machi, Yufu City,
Oita 879-5593, Japan
H. YanoDivision of Radioisotope Research, Department ofResearch Support, Oita University, 1-1 Hasama-machi,
Yufu City, Oita 879-5593, Japan

osteoblastic cells in term of gene expression patternsof various molecular markers and calcium depositionin the cell layer after induction. Furthermore, the
MN16 cells showed strong adhesion to the basic
domain of collagen, a result that is specific for bonederived cells. The MN16 cell line was found to bestably differentiated into bone formation cells in vitro
and should be useful for studying bone biology.
Keywords Bone formation  Cell culture 
MC3T3-E1 cell  Osteoblastic cell line  SV40
large T antigen
Abbreviations
DMEM Dulbecco modified Eagle’s medium
a-MEM Modified Eagle’s medium
SDS
Sodium dodecyl sulfate

Introduction
Bone formation occurs through two different processes: intramembranous ossification and endochondral ossification. The former occurs in craniofacialbones and the lateral part of the clavicles, while thelatter occurs in the long bones, vertebrae, the basal partof the skull and the medial part of the clavicles(Karsenty 2003). Osteoblasts, which play a central rolein both types of ossification, produce a characteristic

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extracellular matrix (ECM) and mineralize the bonematrix. In osteoblasts, collagen, alkaline phosphataseand osteopontin are expressed in the early stages ofdevelopment, while bone sialoprotein (BSP) andosteocalcin are expressed in the late stages (Komori
and Kishimoto 1998; Aubin and Heersche 2002; Choiet al. 1996).
MC3T3-E1 cells, which are derived from newborn
mice calvaria, display a time-dependent and sequential expression of osteoblastic characteristics analogous to those observed in in vivo bone formation(Sudo et al. 1983). Therefore, MC3T3-E1 cells arewidely used as an in vitro model of osteoblastic
development. However, these cells are unstable due toinstability of the mineralization in culture (Baba
2000).
The current study was designed to establish an
osteoblastic cell line with functions such as stableproduction of bone substrate proteins and calcificationin vitro. A mammalian expression vector carrying the
SV 40 large T antigen, an immortalization gene
(Gruenert et al. 1988), was introduced into a primaryculture of cells isolated from the calvaria of newborn
mice.

Materials and methods

Cell Tissue Bank (2014) 15:373–380

Transfection with the SV40 gene and isolationof single clones
Primary cultured cells were grown to confluence in
100-mm dishes and transfected with 5 lg of linearized
DNA of SV40 plasmids using the electroporation
method (Gruenert et al. 1988). The transfected cells
were plated in 60-mm dishes. For cloning, the cells
were replated in a 100-mm dish with a density of
approximately 100 cells/dish. The cells were obtained
from independent colonies. Sixteen single clones wereidentified and characterized for their osteoblastic
phenotype.
Induction of cell calcification
New cloned cells and MC3T3-E1cells (Riken cellbank, Ibaraki, Japan) were plated on 35-mm dishes at adensity of 1 9 105 cell/dish and cultured in a-MEM(Modified Eagle’s Medium) (Life technology Japan,
Tokyo, Japan) and DMEM containing antibiotics and
10 % FBS at 37 °C in 5 % CO2. The next day, after thecells were confirmed to have attached to the plate, theinduction of cells was started. The medium was
replaced with or without 10 mM of b-glycerophosphate and 50 lg/ml of L-ascorbic acid, replaced every
third day (Bellows et al. 1986). The culture wascontinued for 14 and 28 days.

CultureAlizarin red staining
ICR mice were used for the experiment. The animals
were treated in accordance with the Oita University
Guidelines for the Care and Use of Laboratory
Animals based on the National Institutes of Health
Guide for the Care and Use of Laboratory Animals.
Calvaria were removed aseptically from 5 mice (5day-old) and cut into pieces. The pieces were digested
in 5 ml of collagenase (collagenase A, Wako, Japan)
solution (3 mg/ml in Dulbecco modified Eagle’s
medium (DMEM)) (sigma, St. Luis, MO, USA) at
37 °C for 30 min. After the solution was centrifuged at
1,000 rpm for 5 min, the supernatant was discarded.
The pellet was suspended and digested in collagenase
solution at 37 °C for 5 h again. After centrifugation,
the pellet was suspended and cells were plated on three
100-mm dishes and cultured in DMEM containing
antibiotics and 10 % fetal bovine serum (FBS) (Sanko
Junyaku, Tokyo, Japan) at 37 °C in a 5 % CO2.

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After discarding the medium, the cells were washedwith distilled water and fixed with 70 % ethanol. The
fixed cells were washed with distilled water andsubsequently stained with Alizarin red (Sigma-Aldorich, St. Louis, MO, USA) solution for 10 min (Dahl
1952). The remaining dye was washed out with
distilled water.
von Kossa staining
After discarding the medium, the cells were washedwith PBS and fixed with 10 % formalin. The fixed
cells were washed with distilled water three times anddried. The cells were incubated with a 5 % silver
nitrate solution and placed under ultraviolet light until
the staining became dark. Subsequently, they were
washed with distilled water three times and stainedCell Tissue Bank (2014) 15:373–380

with 5 % sodium thiosulfate for 1 min (Puchtler andMeloan 1978).

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Table 1 Primers used for PCR procedures
1. For RT-PCR
SV 40 (J02400)

Quantitative analysis of the Alizarin red staining
A quantitative analysis of the staining for Alizarin red
was performed. The microscopic areas of 10 mm2
were chosen randomly (BZ-9000, Keyence, Osaka,
Japan) and positive areas were analyzed using the BZII analyzing application software program (Keyence).
RT-PCR

Sense

50 -ttaattgtaggctatcaacccg-30

Antisense

50 -ggttaataaggaatatttgatgtatagtgc-30

GAPDH (BC145812)
Sense

50 -agaggtgctgcccagaa-30

Antisense

50 -gtggggagacagaagggaacaga-30

2. For real-time RT-PCR
Osteocalcin (NM007541)
Sense

50 -agactccggcgctacctt-30

Antisense

50 -ctcgtcacaagcagggttaag-30

Alkaline phosphatase (NM007431.2)

Total RNA was extracted from culture cells using
extraction kit (Invitrogen, CA, USA) according to the
manufacture’s instruction. Total RNA (5 lg) was
reverse transcribed using MMLV reverse transcriptase
kit (Invitrogen) with random hexamers at 37 °C for
1 h. The reaction sample was then heated at 95 °C for
5 min to stop reaction. After reverse transcription,
PCR was performed in a 25 ll mixture containing
10 ll of reverse transcription the reaction product,using Go Taq Green Master Mix (Promega, WI, USA).
Thirty-five cycles of denaturation at 95 °C for 1 min,annealing at 55 °C for 1 min and extension at 72 °C
for 1 min were performed for SV40 and GAPDH
genes. The gene-specific primers are listed in Table 1.

Sense

50 -cggatcctgaccaaaaacc-30

Antisense

50 -tcatgatgtccgtggtcaat-30

Col1a1 (NM007742.3)
Sense

50 -catgttcagctttgtggacct-30

Antisense

50 -gcagctgacttcagggatgt-30

Col5a1 (NM 015734.2)
Sense

50 -ctacatccgtgccctggt-30

Antisense

50 -ccagcaccgtcttctggtag-30

Sp7/Osterx (NM 130458.3)
Sense

50 -agagatctgagctgggtagagg-30

Antisense

50 -aagagagagcctggcaagagg-30

RANKL (AF053713.1)
Sense

50 -tgaagacacactacctgactccg-30

Antisense

50 -ccacaatgtgttgcagttcc-30

GAPDH (BC145812)

Real-time RT-PCR
Total RNA (1 lg) was reverse transcribed using
reverse transcriptase (Toyobo, Tokyo, Japan) withrandom hexamers at 30 °C for 10 min and 42 °C for
50 min. The reaction sample was then heated at 94 °C
for 10 min to stop reaction. For a quantitative analysisof the expression level of the mRNA, real-time PCR
was performed using a LightCycler TaqMan Master(Roche, IN, USA). The thermal cycling conditions
included 1 cycle at 95 °C for 10 min, and 40 cycles at
95 °C for 15 s and at 60 °C for 1 min. The relative
mRNA expression level of Osteocalcin, alkaline
phosphatase, Col1a1, Col5a1, Sp7/Osterix andRANKL genes was normalized against that of the
GAPDH gene from the same RNA preparations using
a comparative threshold cycle method (Page andStromberg 2011). The primer sequences are listed inTable 1.

Sense

50 -caatgaatacggctacagcaac-30

Antisense

50 -ttactccttggaggccatgt-30

The accession number is shown in the parentheses

Southern blotting
Genomic DNA was isolated from the culture cells. The
cells were scraped, lysed and digested in a solution
containing proteinase K (500 lg/ml) at 50 °C for 2 h.
After the reaction was completed, the DNA was
precipitated with 0.1 volume of 3 M sodium acetateand 2.5 volumes of 100 % ethanol. DNA was washedwith 70 % ethanol, dried, solved in TE buffer andstored at 4 °C until use.
The DNA was digested with restriction endonuclease, subjected to 0.7 % agarose gel electrophoresis
and transferred to nylon (Nihon Poll Ltd, Tokyo,
Japan) using the methods of Southern blotting. The
filters were hybridized in a solution containing 50 %

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formamide, 59 SSC, 59 Denhardt’s solution (0.1 %polyvinylpyrrolidone, 0.1 % Ficoll, and 0.1 % bovine
serum albumin), 1 % sodium dodecyl sulfate (SDS),
5 % dextran sulfate and 100 lg/ml of salmon sperm
DNA at 65 °C with the 32P-labeled probe, using therandom-primer method. After hybridization was completed, the filters were washed with 0.59 SSC and
0.1 % SDS at 65 °C four times and the bands wereanalyzed with the imaging analyzer BAS2000 (Fujifilm, Tokyo, Japan).
Cell adhesion assay
The recombinant protein of the pro-a3(V)239/371
collagen fragment was generated as previously
described (Puchtler and Meloan 1978). The celladhesion assay was performed as also previously
described (Puchtler and Meloan 1978). In brief, theproteins were put in 24-well plate (2 lg/cm2) at room
temperature for 2 h. The solution was removed and thewell was blocked with 1 % BSA for more than 1 h.
The blocking solution was removed, and the wells
were washed with PBS. The cells were plated in eachwell with a concentration with 1 9 105, and incubate
at 37 °C for 1 h. After washing, attached cells were
fixed with 4 % paraformaldehyde in PBS and stainedwith 0.5 % crystal violate in 10 % ethanol for 30 min.
After washing, cells were solubilized with 1 % SDS,
the absorbance was measured at a wavelength of
595 nm.
Statistical analysis
The values represent the mean ± SD of multiple
independent tests and Student’s t test was used toevaluate the statistical differences between groups,
and a p \ 0.05 was considered to be significant. Eachexperiment was performed at least three times andindividual samples were run in triplicate.

Results
Sixteen murine osteoblastic cell lines were establishedusing cells isolated from the calvaria of 5-day-old
mice. Among these lines, the MN 16 cell line wasselected due to its high expression of osteocalcin andtype I collagen and strong staining with Alizarin red

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Fig. 1 Southern blot analysis. Ten lg of DNA extracted from
MN16 and NIH-3T3 cells were digested with Bam HI (B), Eco
RI (E) and Pst I (P), respectively, run on 0.7 % agarose gel (a),and blotted on the filter and hybridized with the 32P-labeledprobe (b). M DNA size marker. The restriction map of theconstruct DNA, which is integrated in a chromosome, is shownin (c). Dotted lines of both sides are the portions of chromosomeDNA

14 days after differentiation. Therefore, MN 16 cells
were used for further experiments.
A Southern blot analysis was performed to confirm
the integration of the plasmid DNA containing the
SV40 large T antigen gene in the MN 16 cells. The
DNA was digested with restriction endonuclease, Bam
HI, Eco RI and Pst I. The expected fragments, 5. 2, 4.7
and 4.0 kb in size digested with Bam HI, Eco RI and
Pst I, respectively, were seen using the isotope-labeledprobe of the Bam HI/Eco RI fragment derived from the
SV40 large T antigen gene (Fig. 1a–c). Transcripts
derived from the SV40 large T antigen were also
detected during the culture of 28 days with or without
differentiation (data not shown).
The expressions of bone-related genes, including
osteocalcin, alkaline phosphatase, type I and V
collagens, Runx2, Sp7/osterix and RANKL, were
examined using the real-time RT-PCR technique tocharacterize the MN16 cells (Fig. 2). The alkaline
phosphatase level strongly increased after theCell Tissue Bank (2014) 15:373–380

377

Fig. 2 Real time RT-PCR
analysis. The expressions ofalkaline phosphatase (a),osteocalcin (b), typea1(I) collagen (c), typea1(V) collagen (d), Sp7/
Osterix (e) and RANKL(f) 14 and 28 days after
differentiation of MN16 andMC3T3-E1 cells. GAPDH
was used to normalize each
expression level. The valueof the MC3T3-E1 cells
before differentiation was
regarded as 1.0. The data areexpressed as themean ± SD of at least three
independent experiments.
The asterisks indicate
statically significant resultsin the MN16 cells with orwithout induction.
*p \ 0.05. The cells were
cultured in a-MEM

induction (Fig. 2a). Osteocalcin was strongly upregulated in the MN16 cells from day 14 to day 28
after differentiation (Fig. 2b). Two collagen genes, a1
chains of type I and V collagen, were moderately
expressed at the beginning of the culture, then theirlevels gradually decreased during differentiation(Fig. 2c, d). A transcription factor, Sp7/osterix, wasup-regulated during differentiation (Fig. 2e). The
expression of RANKL strongly increased on day 28(Fig. 2f). These experiments were also performed inparallel using MC3T3-E1 cells as controls. In general,the values of the MC3T3-E1 cells were lower thanthose of the MN16 cells. Reverse expression patterns
were also seen in these two cell lines. For example, theexpression of type I collagen was higher under theundifferentiated condition of the MC3T3-E1 cells(Fig. 2c).
Alizarin red and von Kossa staining were used todetect mineralization of nodules in the cultures(Fig. 3). The MN16 cells were strongly stained with

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Fig. 3 Alizarin red (a–h) and von Kossa (i–l) staining. The
MN16 (a, b, e, f, i, j) and MC3T3-E1 cells (c, d, g, h, k, l) were
cultured for 14 days (a–d) and 28 days (e–l) with (a, c, e, g, i, k)or without (b, d, f, h, j, l) induction. The histological images
used are representative of all replicates. The cells were cultured
in a-MEM. Bars 300 lm

Fig. 4 Alizarin red (a–h) staining. The MN16 (a, b, e, f) andMC3T3-E1 cells (c, d, g, h) were cultured for 14 days (a–d) and28 days (e–h) with (a, c, e, g) or without (b, d, f, h) induction.

The histological images used are representative of all replicates.
The cells were cultured in DMEM. Bars 300 lm

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Table 2 Quantitative analysis of the Alizarin red staining
14 days

28 days

MN16
a-MEM

1.00 ± 0.21

0.70 ± 0.17

DMEM
MC3T3-E1

1.07 ± 0.38

0.94 ± 0.08

a-MEM

n.d.

0.57 ± 0.04*

DMEM

n.d.

0.27 ± 0.13*

The value of the MN16 cells on 14 days after differentiation
was regarded to be 1.00. The data are expressed as themean ± SD of three independent experiments. The asterisks
indicate statically significant results in the MC3T3-E1 cells
compared to that of MN16 cells in a-MEM medium on
14 days. * p \ 0.05. n.d. not detectable. The signals were notdetected without differentiation in any experiments (data notshown)

Alizarin red on day 14 and 28 after differentiation(Fig. 3, panels a, e), whereas the MC3T3-E1 cells usedin this experiment were barely stained on day 14 andweakly stained on day 28 (Fig. 3, panels c, g).
Similarly, the MN16 cells were positive for von Kossa
staining on day 28 (Fig. 3, panel i). The cells were also
cultured in DMEM instead of a-MEM. As shown in
Fig. 4, the staining patterns using Alizarin red in
DMEM were similar to those in a-MEM. The
qualitative analysis of Alizarin red staining by
microscopy confirmed that the mineralization on day
14 and 28 was more prominent in the MN16 cells
compared to the of MC3T3-E1 cells (Table 2).
A previous study showed that the basic segment
derived from pro-a3(V) collagen preferentiallyadheres to rat ROS 17/2.8 sarcoma cells but not to
rat RCS chondrosarcoma cells (Yamaguchi et al.
2005). In this experiment, it strongly adhered to MN16
and MC3T3-E1 cells and barely to rat RCS chondrosarcoma cells (Fig. 5).

Discussion
Various primary cells and cell lines have been
employed for studying osteoblast cell biology. The
primary cultures show the characteristic features ofosteoblasts in vitro. However, they can undergo
limited passages and sometimes show a heterogeneous
phenotype due to the tissue source of tissues and/or
method of preparation. To overcome these disadvantages, immortalized cell lines have been developed.

Fig. 5 Cell adhesion assay. MN16, MC3T3-E1 and RCS cells
were plated on dishes coated with the recombinant protein of
pro-a3(V) collagen polypeptide. The values shown are themean ± SD of three experiments. The asterisks indicate
statically significant results between the cells. *p \ 0.05. The
cells were cultured in a-MEM

The most commonly used cell lines are SaOs-2, MG63 and MC3T3-E1 (Czekanska et al. 2012). The SaOs2 cell line was derived from a human sarcoma, anddoes not show the whole range of osteoblast phenotype changes. The MG-63 cell line derived from ahuman juxtacortical osteosarcoma arrests in the preosteoblastic state. The MC3T3-E1, preosteoblastic
murine cell line, was originally established fromnewborn calvaria based on high alkaline activity in theresting state (Sudo et al. 1983). However, this cell line
sometimes appears to be unstable due to a loss ofability to achieve mineral deposition (Baba 2000).
Therefore, this study aimed to establish a cell line withosteoblastic characteristics that is stably expressedin vitro. The MN16 cell line was selected on the basisof a high expression of osteocalcin and type I collagenand positive staining with Alizarin red 14 days after
differentiation. The cell line shows the characteristics
of osteoblastic cells in terms of gene expression ofvarious molecular markers for osteoblast and calcium
deposition in the cell layer after induction. However,alkaline phosphatase, an early marker, and osteocalcin, a late marker showed a similar expression patternin the experiment. Since the MN16 cell line wasselected as a clone with a high expression of osteocalcin after induction, the parental cell from which the
MN16 cell line was derived might has had characteristics of preosteoblasts before transfection. Therefore,

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osteocalcin might have been expressed rather earlier inthe MN16 cell line.
For osteoblastic cells, a-MEM is the standard
medium to promote the cell growth and survival(Hinoi et al. 2002). However, The MN16 cells could
be cultured in DMEM and thus demonstrated similar
characteristics to those cells (Fig. 4). Each gene
expression examined was higher in the MN16 cellline than in the MC3T3-E1 cell line overall. The
MN16 cell line shows a stable and reproducible
expression pattern, and is superior to the MC3T3-E1
cell line in term of its osteoblastic characteristics. In
conclusion, the data described above showed that the
MN16 cell line exhibits immature osteoblastic characteristics and can be differentiated to osteoblast inwhich calcium deposition occur in the cell layer
similar to bone matrix in vivo. Therefore, this cell line
should be an excellent tool for studying bone biology.
Acknowledgments We thank Dr. M. Watanabe, Mr. S. Imi, Ms
A. Yasuda and the staff members of Divisions of Life science
Research, Laboratory Animal Science and Radioisotope Research,
Department of Research Support, Research Promotion Project,
Oita University. This work was supported by Grants-In-Aid for
Scientific Research (No. 20390402 to H.Y.) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan.
Conflict of interest The authors report no conflicts of interest.

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