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Jpn. J. Oral Biol.,23: 899-901, 1981.
Establishment of a clonal osteogenic cell line from newborn
mouse calvaria
Hiro-aki Kodama, Yuji Amagai, Hiroko Sudo*, Shiro Kasai and Shigehisa Yamamoto
Department of Physiology(Chief: Prof . Shiro Kasai) and Second Department of Anatomy*
(Chief: Prof. Shigehisa Yamamoto),Tohoku Dental University 31-1 Misunzido,
Tomitamachi, Koriyama, Fukushinza 963, Japan
•kAccepted for publication December, 20, 1981•l
Key words: cell culture/osteogenic cell line/osteoblast/alkaline phosphatase/ calcification
Although there are abundant studies onmetabolism and hormonal responsiveness ofisolated bone cells in culture , only a fewreports have demonstrated calcified tissueformation in bone cell culture1,2).An osteo-genic cell line that retains the capacity todifferentiate into osteoblasts and mineralizein vitro would be a very useful model systemfor the study of differentiation in the osteo-blast series and the mechanism of calcifica-tion. The present studies aimed to establishcell lines from newborn mouse calvaria undera rigid 3T3 subculturing schedule'); i . e.,cells were transferred at 3-day intervals ata relatively low inoculum of 3 x 105 cells/25cm2 flask in order to select cells sensitive to
postconfluence inhibition of division. Fur-ther selection was made for those cells ex-hibiting alkaline phosphatase (ALP) activityas a marker for osteoblasts4,5) , and such linewas examined for the capacity to mineralizein vitro.
Calvaria were aseptically removed from 9newborn C57BL/6 mice (within 24hr afterbirth). After stripping cartilage and peri-osteal tissues and washing with Cal+ , Mg2+free phosphate-buffered saline (PBS-) , bones
were finely minced with scissors . The bonefragments were plated in eight 60 mmPetri dishes with a-MEM(Flow Lab .)sup-plemented with 10 % fetal bovine serum(Flow Lab.). The cultures were incubatedat 37°C in a fully-humidified atmosphere of5 % CO2 in air. Cells which migrated fromthe fragments were allowed to grow for 6days. These cultures were independentlysubcultured using 0.05% trypsin(GIBCO)
plus 0.02% EDTA in PBS- and put on a
rigid transfer schedule following a slight
modification of the method of Tadaro and
Green3), in which every three days cells
were transferred to 5 ml of fresh medium
in new 25 cm2 plastic flasks (Corning) at
3•~105 cells/flask. Cumulative growth curves
of four such cultures are shown in Fig.1 .
Severe reduction in the growth rate was
observed only in the MC3T3-E culture from
the 9 th to the 15 th passage, and then the
cells began to grow actively. There was
little difficulty in maintaining the other cul-
tures, and all cultures developed into per-
manent cell lines.
Each cell line was assayed for ALP ac-
tivity at 3-day intervals after plating 3•~105
cells/flask. As shown in Fig.2, growing
MC3T3-E cells showed very low ALP ac-
tivity. After the cells became confluent on
day 3-4, however, the activity began to
increase; and by day 18, it reached 80
timoles/hr/mg protein. ALP activity was
also detected in the cells of the other lines,
but its maximum was less than 15 pmoles/
hr/mg protein. These results strongly sug-
gested that MC3T3-E cells were an osteo-
genic cell line. Therefore, cells from the
50th passage were inoculated at 50 cells/60
mm plastic dish (Falcon); and 17 days later,
colonies were stained for ALP activity as
described by Maio and De Carli6). Ten
ALP-positive colonies (stained yellow) were
isolated, and each clone was examined for
ALP activity. A clone, MC3T3-E1, showing
the highest ALP activity on day 15 of cul-
ture, was chosen and used for subsequent
900Jpn. J. Oral Biol.,23: 899-901, 1981
Fig.1 Cumulative growth curves of cultures
of newborn mouse calvaria cells. At
each transfer, the cell number in
each culture was counted and cumu-
lated, taking the cell number at the
start of the 3T3 subculture schedule
as 1. A, MC3T3-E culture; 0,
MC3T3-G1 culture;•,,MMC3T3-G2
culture; MC3T3-H2 culture.
studies.
The cells were cultured with a-MEM sup-
plemented with 10% newborn calf serum
(Flow Lab.) and subcultured twice a week
using 0.001% pronase E(Kaken Kagaku)
plus 0.02% EDTA in PBS-. For experi-
ments, cells were plated at 5•~104 cells/35
mm plastic dish(Falcon) in 1.5 ml of medium
and refed every three days. On day 30,
cultures were fixed and stained. MC3T3-
El cells grew to form multiple cell layers
in contrast to other 3T3 lines3). By azan
staining, the intercellular spaces were stained
with aniline blue, and the fibrous nature of
this material became evident (Fig.3a). In
MC3T3-E1 cultures, the cells were highly
piled up in many localized areas. Cells in
these areas showed intense ALP activity
(Fig.3b), and the intercellular spaces among
these cells were stained with alizarin red S
(Fig.3c). Recently, hydroxyapatite crystal
deposition on a collagen-rich ground sub-
stance in the intercellular spaces was de-
monstrated in day 30 cultures by electron-
microscopic observations, energy dispersive
X-ray analysis, as well as electron diffrac-
tion analysis. Active production of matrix
vesicles by osteoblast-like cells and initial
mineral deposition in these vesicles were
also observed(H. Sudo et al., manuscript
Fig.2 ALP activity of MC3T3-E cells. MC3T3-E cells were inoculated
at3•~105 cells/25 cm2 flask and refed every three days. At the
indicated times, cells were washed three times with HEPES-buf-
fered saline (0.15 M NaC1, 5 mM HEPES, pH7.4), harvested and
assayed for ALP activity as described by Koyama and Ono".
Protein was determined by the method of Lowry et al.8).
H. Kodama, et al.: Establishment of a clonal osteogenic cell line 901
Fig.3 Photomicrographs of histochemically stained MC3T3-E1 cells. 30-day-old MC3T3-E1 cul-
tures were fixed with 2.5% glutaraldehyde for 2 hr and histochemically stained. ALP
activity was detected by the method of Burstone9). a, Azan staining;b, staining for
ALP activity; c, alizarin red S staining for calcium(magnification•~150).
in preparation). These results indicate thatthe clonal osteogenic cell line, MC3T3-E1,has the capacity to produce collagen anddifferentiate into osteoblast-like cells, form-ing calcified tissue in vitro similarly to theirin vivo counterparts.
Acknowledgement
We wish to thank Drs. H. Koyama, M. Kume-gawa, and R. Sukekawa for valuable discussionand encouragement, and Miss M. Kageyama andMrs. C. Endo for technical assistance.
Reference
1) Binderman, I., Duksin , D., Harell, A., Katzir,E. and Sachs, L.: Formation of bone tissuein culture from isolated bone cells . J. CellBiol., 61: 427-439, 1974.
2) Williams, D. C., Boder, G. B., Toomy, R. E.,Paul, D. C., Hillman, C. C. Jr., King, K. L.,van Frank, R. M. and Johnston , C. C. Jr.:Mineralization and metabolic response in se-rially passaged adult rat bone cells. Calcif.Tissue Int., 30: 233-246, 1980.
3) Todaro, G. J. and Green, H.: Quantitative
studies of the growth of mouse embryo cellsin culture and their development into estab-lished line. J. Cell Biol., 17: 299-313, 1963.
4) Doty, S. B. and Schofield, B. H.: Enzymehistochemistry of bone and cartilage cells.Prog. Histochem. Cytochem., 8: 1-38, 1976.
5) Bernard, G. W.: Ultrastructural localizationof alkaline phosphatase in initial intramem-branous osteogenesis. Clin. Orthop., 135:218-225, 1978.
6) Maio, J. J. and de Carli, L. L.: Distribu-tion of alkaline phosphatase variants in aheteroploid strain of human cells in tissueculture. Nature, 196: 600-601, 1962.
7) Koyama, H. and Ono, T.: Further studieson the induction of alkaline phosphatase by5-bromodeoxyuridine in a hybrid line betweenmouse and chinese hamster in culture. Bio-chim. Biophys. Acta, 264: 497-507, 1972.
8) Lowry, O. H., Rosebrough, N. J., Farr, A. L.and Randall, R. J.: Protein measurementwith the Folin phenol reagent. J. Biol.Chem., 193: 265-275, 1951.
9) Burstone, M. S.: Hydrolytic enzyme in den-tinogenesis and osteogenesis. Amer. Assoc .Advance Sci., 64: 213-217, 1960.
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