JPH074225B2 - Culture bag - Google Patents

Description

TECHNICAL FIELD The present invention relates to a culture bag used for culturing cells such as animal tissues.

(Prior Art) When culturing animal tissue or the like, conventionally, cells and a culture solution are put into a petri dish or a flask, and this is placed in a carbon dioxide gas incubator, and sterilized air / carbon dioxide gas is placed in this incubator. The mixed gas (generally 5% by volume of carbon dioxide) was introduced, and the cells and the culture solution were placed under this mixed gas atmosphere.

The method of placing the cells and the culture solution in this mixed gas atmosphere is
A small gap is formed in the petri dish or flask so that gas can pass therethrough, and the mixed gas in the carbon dioxide incubator is put into the dish or flask through the gap and brought into contact with the culture solution.

(Problems to be Solved by the Invention) Since the mixed gas in the carbon dioxide incubator has been sterilized in advance, even if the mixed gas is brought into contact with the culture medium, the culture medium is not contaminated with various bacteria. However, when the door of the carbon dioxide incubator is opened, bacteria in the air are taken into the incubator,
It comes into contact with the culture solution and causes the inconvenience of contaminating the culture solution.

Therefore, it is considered to attach a sterilizing filter to the cap mouth of a flask or the like, but in this case, the filter mounting area is limited and it is not suitable for mass culture.

The present invention has been made in view of such circumstances, and an object thereof is to provide a culture bag in which a gas such as carbon dioxide can be circulated and the gas permeation area per unit volume can be freely controlled.

(Means for Solving the Problems) The culture bag according to the present invention comprises 100 parts by weight of vinyl chloride resin or a copolymer resin of vinyl chloride and a monomer copolymerizable therewith, ethylene / n-butyl acrylate / At least one opening is formed in a container body formed by fusion-bonding a sheet material having a thickness of 0.1 to 0.5 mm, which is composed of 150 to 260 parts by weight of a carbon monoxide copolymer.

When cells are cultured in a culture medium, oxygen gas is required for the cells to grow. In addition, PH in the culture system is about 7.
Carbon dioxide is required to control to 4-7.6. If this PH exceeds 7.8, culture failure will occur. Polymeric resins are the most suitable as a material that allows these gases to freely ventilate and store the culture solution. Also, the items required for the material constituting the container are a transparent film in order to check the growth degree of cells with a microscope, etc., since it is necessary to have sealing property in addition to the above, fusion bonding is possible. Etc. are required.

Materials satisfying these requirements include vinyl chloride resins or copolymer resins of vinyl chloride and a monomer copolymerizable therewith (for example, vinyl chloride / ethylene copolymer, vinyl chloride / propylene copolymer, vinyl chloride). A sheet material composed of 100 parts by weight of vinyl acetate copolymer) and 150 to 260 parts by weight of ethylene / n-butyl acrylate / carbon monoxide copolymer is used.

Further, the thickness of the sheet-shaped material is such that it has gas permeability and does not cause the container to burst when filled with the content liquid. It is preferably 0.1 to 0.5 mm. If it exceeds 0.5 mm, the transparency of the sheet will be poor and the inspection of the contents will be hindered. Furthermore, in the container body that becomes like this,
At least one opening is required. This opening is used for introducing the culture solution, discharging the solution, introducing the washing solution, introducing the cells,
It is used for collecting cultured cells or sampling during culture. The openings may be provided separately depending on the purpose of use. This opening can be provided by attaching to the container body a tube made of the same material as the container body (for example, a tube having an inner diameter of 3 to 5Φ). Since the container body, the tube, and the like can be fusion-bonded by high-frequency fusion, heat fusion, or the like, complete hermeticity is maintained.

The bag for culture of the present invention is used, for example, as follows. After being sterilized with ethylene oxide gas (may be sold in a sterilized bag after sterilization), culture solution and cells are introduced into the container body from the opening in the clean bench, and opened with a pinch cock etc. The part is sealed. This is used by statically culturing in a gas atmosphere necessary for culturing, and then removing the culture solution and cells from the opening.

In the present invention, instead of closing the above-mentioned opening with a pinch cock or the like, a sterilized cap or a rubber stopper may be attached to the opening. Further, for sampling during culturing, a tube having a film formed inside or a tube having a mixed injection rubber button fitted therein may be attached to the container body in addition to the above-mentioned opening.

(Function) The container body is composed of 100 parts by weight of vinyl chloride resin or a copolymer resin of vinyl chloride and a monomer copolymerizable therewith, ethylene / n-butyl acrylate / carbon monoxide copolymer 15
Since it is made by fusing a sheet material with a thickness of 0.1 to 0.5 mm consisting of 0 to 260 parts by weight, it is possible to observe the contents while allowing gas to permeate through this sheet material. it can.

(Example) 100 parts by weight of vinyl chloride / ethylene copolymer (average degree of polymerization 1300, ethylene content 4% by weight) and ethylene / n-butyl acrylate / carbon monoxide copolymer (n-butyl acrylate content 30) % By weight, carbon monoxide content 10% by weight) 160 parts by weight, Ca-Zn-based stabilizer (Mark 37 manufactured by ADEKA ARGUS Co., Ltd.) 1.0 part by weight, epoxidized soybean oil (O-130P manufactured by ADEKA ARGUS Co., Ltd.) 15 By weight, 1 part by weight of polyethylene wax (high wax 4202E manufactured by Mitsui Petrochemical Co., Ltd.) was melt-kneaded with an extruder (kneading temperature 180 ° C.), and the obtained vinyl chloride resin composition was pelletized.

Then, a T-die having a width of 400 mm was attached to a 65 mm single-screw extruder, and the sheet material was extruded by molding at 180 ° C. Also,
The tube was molded with the same extruder. Then, the sheet-shaped material and the tube were fusion-bonded to obtain a culture bag 1 as shown in FIG.

The obtained culture bag 1 has an inner volume of 4 ml and an outer diameter of 5 as an opening 10 in a container body 2 having an inner volume of 200 ml and a sheet thickness of 0.15 mm.
mm culture medium introduction tube 11, cell introduction tube 12
And the sampling tube 13 was formed.

In addition, the sheet thickness of the container body 2 is 0.4 mm by the same method.
To obtain a culture bag 1.

Next, an example of an experiment carried out using the culture bag 1 thus obtained will be described.

[First Example] 140 ml in the above culture bag sterilized with ethylene oxide
Serum-free medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) was added, and cells were added thereto at 5 × 10 ⁴ cells / ml, and the mixture was incubated at 37 ° C. for 5
The cells were statically cultivated for 5 days in a carbon dioxide incubator under a% carbon dioxide atmosphere. Then, the number of cells on the 5th day of culture was measured. In addition, the total light transmittance of the culture bag is JIS (JIS K7105).
It was measured based on. The results are shown in Table 1.

The cells are P3 / NS1 / 1-Ag4-1 cells (original ATCC strain number T
IB-18 Purchased from Dainippon Pharmaceutical Co., Ltd. Below, cell line NS-1
And And a hybridoma strain obtained by cell fusion using mouse spleen cells.

In addition, as a comparative control, 5 of the above hybridoma strain was added to a culture flask (150 cm ² purchased from Iwaki Glass Co., Ltd.).
The cells were added at a concentration of × 10 ⁴ cells / ml, and a serum-free medium was added to bring the total volume to 30 ml, followed by static culture for 5 days in a carbon dioxide incubator under the same conditions. At that time, the lid of the culture flask was slightly opened. Then, the number of cells on the 5th day of culture was measured. The results are shown in Table 1.

[Second Experimental Example] RPMI1640 medium (purchased from Dainippon Pharmaceutical Co., Ltd.) was prepared aseptically by adding 10% fetal bovine serum to a container body with a thickness of 1 cm (about 140 ml). As described above, the cells were placed in a culture bag (sterilized with ethylene oxide). Add cell line NS-1 to this to give 5 × 10 ⁴ cells / ml.
In a carbon dioxide incubator at 7 ° C and 5% carbon dioxide gas atmosphere, 5
The culture was allowed to stand for a day. Then, the number of cells on the 5th day of culture was measured. The results are shown in Table 2.

As a comparative control, culture cell (150 cm ² purchased from Iwaki Glass Co., Ltd.) was charged with 5 × 10 ⁴ Cel cell line NS-1.
The culture medium was added at ls / ml and the same medium as above was added to bring the total volume to 30 ml, followed by static culture for 5 days in a carbon dioxide incubator under the same conditions. At this time, the lid of the culture flask was slightly opened. Then, the number of cells on the 5th day of culture was measured. The results are shown in Table 2.

(Effects of the Invention) As described above, according to the present invention, while gas permeates through the sheet-shaped material constituting the culture bag,
Since it is possible to observe the contents, it is possible to carry out a closed-state culture, and it is possible to prevent contamination of the culture with bacteria. In addition, since the container body is formed by fusion bonding, the container body can be easily made to have an arbitrary size. further,
Since the sheet material itself has gas permeability, the amount of gas permeation can be freely controlled.

[Brief description of drawings]

FIG. 1 is a plan view showing the outline of the overall structure of a culture bag according to the present invention. 1 …… Culturing bag 10 …… Opening 11 …… Culture fluid introduction tube 12 …… Cell introduction tube 13 …… Sampling tube 2 …… Container body

Claims (1)

[Claims] 1. An ethylene / n-butyl acrylate / carbon monoxide copolymer, and 100 parts by weight of a vinyl chloride resin or a copolymer resin of vinyl chloride and a monomer copolymerizable therewith.
A culture bag characterized in that at least one opening is formed in a container body formed by fusion-bonding a sheet material having a thickness of 0.1 to 0.5 mm consisting of 0 to 260 parts by weight.