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

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