Professional Documents
Culture Documents
USOO5225346A
[11]
[45]
Matsumiya et al.
[54] CULTURE BAG
[75] Inventors: Toshiliaru Matsumiya, Yamaguchi'
Shozo Shiraishi, Kanagawa; Kazuo
4,939,151 7/1990
4,968,624 11/1990
2435524
WO90/O3427
Japan
2-7027e[u]
[51]
[52]
[58]
[56]
Brewer ...... ..
4,135,026
4,698,372 10/1987
4,910,147
Moss .................. ..
France .
1986).
3,184,395
4/1980
1990).
Brewer .................. ..
8/1963
3,102,082
Jul. 6, 1993
OTHER PUBLICATIONS
[30]
5,225,346
Japan
[22] Filed:
Patent Number:
Date of Patent:
435/301
435/299
525/61
.. 521/149
435/284
US. Patent
July 6, 1993
sheet 1 of 3
5,225,346
F|G.2
16 1 1171 18
11\
12
13
12
19
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US. Patent
July 6,1993
Sheet 2 of 3
5,225,346 '
US. Patent
FIG.8
July 6, 1993
Sheet 3 or 3
FIG.9
5,225,346
5,225,346
CULTURE BAG
above-mentioned object.
dioxide.
The second aspect of the present invention is embod
ied in a culture bag which comprises an integrally
cells.
combined with one another to form a laminate, if neces 40 for the storage room being made of a resin composition
sary.
coextrusion from any of (a) ethylene-a-ole?n (C440) 50 by fusion-bonding the sheet for the culture room (de
?ned above) and a medium storage room formed by
copolymer having a density of about 0.91-0.925 g/cln3,
fusion-bonding the sheet for the storage room (defuied
(b) ethylene-methacrylic acid copolymer, (c) an iono
above), said two rooms communicating with each other
mer, and (d) ionomer/polyester elastomer and linear
The containers proposed in the above-mentioned 55 formed in the culture room and at least one opening
bags.
Japanese Patent Publication No. 50063/1980 (US
Pat. No 3,780,140) discloses a resin composition from
which to produce ?exible plastic containers. This resin
5,225,346
25
INVENTION
butyl acrylate-carbon monoxide copolymer, together
The culture bag pertaining to the ?rst aspect of the
with optional additives including a stabilizer (such as
present invention comprises an airtight culture room
calcium stearate and zinc stearate), a lubricant (such as
and at least one opening formed therein, said culture 35 polyethylene wax and stearic acid), and the like. The
room being formed by fusion-bonding a transparent
blending may be accomplished by using a batchwise
sheet for the culture room having speci?c properties.
mixer (such as roll and mixer) or a continuous mixer
Oxygen is necessary for cells to grow in a medium
(such as twin-screw extruder).
and carbon dioxide is necessary for a medium to be in
The sheet for the culture room may be produced by
the pH range 7.4-7.6. At a pH higher than 7.8, cell
extruding the resin composition in pellet form. The
growth does not proceed smoothly. Therefore, it is
sheet should be 0.1-0.5 mm thick, preferably 0.15-0.4
necessary that the culture bag be made of a material
having a good gas permeability to oxygen and carbon
dioxide. In addition, the material for culture bags should
has a gas permeability of 600-3000 ml/m2.24hr.atm, 55 culture room is made. This tube may be produced by
preferably' 800-2600 ml/m2.24hr.atm, to oxygen and a
extruding the resin composition in pellet form.
gas permeability of 1000-30,000 ml/m2.24hr.atm, pref
The culture bag of the present invention may be
erably l50025,000 ml/m2.24hr.atm, to carbon dioxide.
formed from two pieces of the sheet for the culture
If the amount of ethylene-n-butyl acrylate-carbon mon
room cut in approximate rectangle of desired size from
oxide copolymer is less than speci?ed above, the result
the web. The two sheets are placed one over the other,
ing sheet is poor in clarity and ?exibility; in the opposite
with a tube for the opening held between them at one
case, the resulting sheet is poor in strength and liable to
end thereof, and the peripheries of the sheets are fusion
blocking. The sheet for the culture room should have a
bonded together with the tube by high-frequency seal
total light transmittance of 80l00%.
ing or heat sealing which makes the culture bag com
The vinyl chloride homopolymer or copolymer men 65 pletely airtight.
tioned above should be one which has an average de
The culture bag of the present invention is used in the
gree of polymerization of 300-5000, preferably
following manner. First, the culture bag is sterilized by
800-2000, as measured according to JIS K6721. The
ethylene oxide. The sterilized culture bag (with or with
5,225,346
and cells are injected into the culture room through the
sheet for the storage room is formed from a resin com 50 ture. Finally, the medium is discharged through the
bility.
5,225,346
may be formed from one piece of the sheet for the stor
same manner as used for the above-mentioned sheet for
age room and one piece of a sheet which is not origi
the culture room.
nally intended for the storage room. The sheet which is 55
The laminated sheet may be prepared by one of the
not originally intended for the culture room and the
following four methods. (a) Coating both sides of a core
sheet which is not originally intended for the storage
sheet with a solution of resin composition which forms
room may be produced from polyvinyl chloride, ethy
a ?lm after drying. Or, dry bonding of a core sheet with
lene-vinyl acetate-carbon monoxide copolymer, or
two sheets as the outer layers. (b) Thermolamination of
ethylene-vinyl acetate copolymer (containing 40-50 wt. 60 a core sheet with two sheets as the outer layers. (0)
% vinyl acetate), or a mixture thereof. These sheets
Coating both sides of a core sheet with an extruded
should have a degree of gas permeability equal to or
molten resin which forms the outer layers. (d) Lamina
lower than that of the sheets for the culture room and
tion of a core sheet and outer layers by coextrusion.
storage room. In the case of the second and third types
The thus obtained laminated sheet is superior in gas
of the culture bags, the tube as the passage should pref 65 permeability and free of plasticizer such as dioctyl
erably be provided with a stopper which can be opened
phthalate. In addition, it is characterized by ?exibility,
as fusion-bonding.
5,225,346
The invention will be described in more detail with
10
REFERENTIAL EXAMPLE 1
An ethylene-n-butyl acrylate-carbon monoxide co
'
Experiment 1
Experiment 2
tube die.
Cell culture was carried out in the same manner as in
The thus obtained sheet (for the culture room) was 45 Experiment 1, except that the culture medium (140 ml)
cut into two approximate rectangles measuring 20 cm
was serum-free medium RPMI 1640 (purchased from
by 27 cm. The two rectangular sheets were placed one
Dainippon Pharmaceutical Co., Ltd.) incorporated
over the other, with three pieces of the tube held be
with 10 vol % fetal calf serum under sterile conditions.
tween them at one end thereof. The peripheries of the
The cells were NS-l as many as 5 X 104 cells/ml. At the
sheets were fusion-bonded, together with the tubes, by 50 end of cell culture, the number of cells was counted.
high-frequency sealing. Thus there was obtained a cul
For comparison, the same procedure as above was
ture bag (1) consisting of a 200-ml culture room (2), a
repeated using a culture ?ask as in Experiment 1 that
medium injection tube (3), a cell injection tube (4), and
employed 30 ml of culture medium. The cells were
a sampling tube (5), as shown in FIG. 1.
NS-l as many as 5X10 cells/ml. At the end of cell
55
culture,
the number of cells was counted.
EXAMPLES 2 TO 4
The results are shown in Table 1.
Culture bags were. prepared in the same manner as in
It is noted from Table 1 that the culture bags in Ex
Example 1 except that the amount of ethylene-n-butyl
amples 1 to 4 are superior in gas permeability and clarity
acrylate-carbon monoxide copolymer in the resin com
and they permit a high growth rate of cells.
TABLE 1
Example
No.
Copolymer
(parts by
weight)
Thickness
of sheet
(mm)
Total light
transmittance (%)
Oxygen
permeability
Carbon dioxide
permeability
(ml/m2 24 hr - atm)
(ml/m2 24 hr - atm)
Cell count
on 0th day
(cells/ml)
Cell count
on 5th day
Experiment 1
Cell count
on 5th day
Experiment 2
160
0.15
93.4
1,680
12,430
s x 104
8.0 x 105
8.0 x 105
2
3
4
240
160
240
0.15
0.4
04
93.7
91.8
92.1
2,460
960
1,040
20,260
5,800
7,200
5 X 104
5 X 104
5 x 10
8.6 X 105
7.0 X 105
7.3 x 105
5,225,346.
11
12
TABLE l-continued
Oxygen
Example
No.
Copolymer
(pans by
weight)
Thickness
of sheet
(mm)
Total light
transmittance (%)
permeability
(ml/mz 24 hr - atm)
Culture
Carbon dioxide
permeability
(ml/m2 ~
24 hr - atm)
Cell count
on 0th day
(cells/ml)
Cell count
on 5th day
Experiment 1
Cell count
on 5th day
Experiment 2
5 >< 104
6.5 x 105
7.0 X 105
?ask
EXAMPLE 5
EXPERIMENT 3
The culture bag obtained in Example 5 was used for
experiments on cell culture in the following manner.
gles. This sheet has an oxygen permeability of 170 20 free medium (RPMI 1640, the same one as used in Ex
ml/m2.24hr.atm and a carbon dioxide permeability of
periment 2) through the medium injection tube. With
890 ml/m2.24hr.atm. This sheet is the under-mentioned
the medium injection tube closed by a pinchcock at its
sheet (12) for the storage room.
end, the culture bag was kept in an incubator of 5%
The resin composition prepared in Example 1 was
carbon dioxide atmosphere at 37 C. for 16 hours. Then,
formed by extrusion into a 0.2-mm thick sheet for the 25 the connecting tube was opened to transfer the culture
culture room. This sheet was cut into approximate rec
EXAMPLE 6
two sheets (12 and 12) and the culture room is formed
by one each of sheet (12) and sheet (13). These three
40
5,225,346
13
14
injection tube (3), the cell injection tube (4), and the
sampling tube (5) at its upper end.
The culture bags shown in FIGS. 7 to 9 permit grown
EXAMPLE 8
EXPERIMENT 4
The culture bag obtained in Example 6 was used for
experiments on cell culture in the following manner.
the medium injection tube closed by a pinchcock at its 20 is 10 parts by weight. The core (0.25 mm thick) was
end, the culture bag was kept in an incubator of 5%
prepared from the same ethylene-n-butyl acrylate-car
carbon dioxide atmosphere at 37 C. for 16 hours. Then,
bon monoxide copolymer as mentioned above. This
the connecting tube was opened to transfer the culture
laminated sheet was cut into two rectangular sheets,
medium into the culture room, and as many cells as
which were then formed into the culture bag in the
5 X 104 cells/ml were immediately injected into the cul 25 same manner as in Example 1.
ture room through the cell injection tube. Cell culture
EXAMPLE 9
was carried out under the same conditions as in Experi
ment 1, and the number of cells was counted on the
The same procedure as in Example 8 was repeated
fourth day of culture. It was found that the number of
except the following changes. The ?rst outer layer (72),
cells increased from 5 X 104 cells/ml to 8.0>< 105 cells/ml 30 0.08 mm thick, was prepared from a vinyl chloride type
EXAMPLE 7
Cell culture was carried out using the culture bag (41)
COMPARATIVE EXAMPLE 1
the right side, forming the gap (54) through which cells
pass.
45
Thickness
of sheet
Total light
transmit-
Oxygen
permeability
Carbon dioxide
permeability
Cell countr
on 0th day
Cell count
on 4th day
No.
(mm)
tance (%)
(ml/m2 - 24 hr - atm)
(ml/m2 - 24 hr ~ atm)
(cell/ml)
(cell/ml)
a
9
Comparative
0.35
0.37
0.36
92.6
92.1
93.8
2.200
1,400
500
12,000
7,000
3,500
5 X 10
5 x 10
5 X 104
9.0 x 105
8.8 x 10 5
5.8 X 10 5
s x 10
6.3 x 10 5
. Example 1
Culture
5,225,346
15
16
TABLE 2-continued
Thickness
Total light
Cell countr
of sheet
transmit-
Oxygen
permeability
Carbon dioxide
Exmaple
permeability
on 0th day
Cell count
on 4th day
No.
(mm)
tance we)
(ml/m2 - 24 hr - atm)
(ml/m2 - 24 hr - atm)
(cell/ml)
(cell/ml)
?ask
least one each of cell injection opening and medium 25 homopolymer or copolymer and 30-150 parts by
discharge opening formed in the culture room and a
weight of ethylene-vinyl acetate-carbon monoxide co
medium injection opening formed in the medium stor
polymer, and having a gas permeability of 100-350
age room, with the culture room and medium storage
ml/m2.24hr.atm to oxygen and a gas permeability of
room communicating with each other through a pas
200-1000 ml/m2.24hr.atm to carbon dioxide.
sage, said culture bag being formed by fusion-bonding
5. A culture bag as claimed in claim 4, wherein the
the peripheries of two sheets for the storage room and
culture room is formed by fusion-bonding the peripher
one sheet for the culture room in such a manner that the
ies of two sheets, at least one of which being made of
two sheets for the storage room face to each other to
said resin composition.
form the medium storage room and one of the two
sheets for the storage room and the one sheet for the
culture room face to each other to form the culture
room, said sheets for the storage room being made of a
part.
sheet for the culture room, and said core being made of
55
65