JPH0669361B2 - Rubber parts for cell or microorganism culture equipment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION a. Field of Industrial Application The present invention relates to a rubber for culture equipment that is non-toxic to cells or microorganisms and is suitable for various rubber parts used in a portion of a culture equipment for cells or microorganisms in contact with a medium. Regarding parts.

b. Conventional technology In recent years, along with the development of cell or microorganism culturing technology, bacteria, animal cells, plant cells, etc. have been cultured in large amounts, and large quantities of useful substances are produced by these cells or microorganisms. It has become to. In order to efficiently cultivate these cells or microorganisms, it is important to select a culture device suitable for culturing, and in particular, the parts used in contact with the medium are made of materials that are less toxic to the cells or microorganisms. The use of culture equipment is an important point that influences the culture efficiency.

Among the parts conventionally used in the part of the culture device that contacts the culture medium, parts such as a tube for a tube pump, an O-ring, and packing that are required to have rubber elasticity include, for example, vinyl chloride to which a plasticizer is added. Parts made up of were known.

c. Problems to be Solved by the Invention However, since the rubber parts for cell culture equipment conventionally used for the part in contact with the culture medium have toxicity to cells or microorganisms, the survival rate of the cells or microorganisms during culture is increased. However, there was a drawback that the culture efficiency was lowered as a result. Therefore, there has been a demand for the appearance of a rubber component for cell or microorganism culture equipment that is non-toxic to cells or microorganisms.

d. Means for solving the problems The present inventors have conducted intensive research to solve the above-mentioned drawbacks,
It was found that a rubber part formed by a crosslinked product of a tetrafluoroethylene-propylene copolymer is nontoxic to cells or microorganisms, and the present invention has been completed based on this finding.

That is, the present invention provides a rubber component for cell or microorganism culture equipment (hereinafter, simply referred to as “rubber component”), which is characterized by being formed by a crosslinked product of a tetrafluoroethylene-propylene-based copolymer. It is also.

The tetrafluoroethylene-propylene-based copolymer used in the present invention is a copolymer in which tetrafluoroethylene and propylene are arranged almost alternately from the monomer reactivity and has the following general formula: [N and m represent integers. ] It has a structure shown in, and is commercially available under the trade name Afras (manufactured by Asahi Glass Co., Ltd.).

In the present invention, in order to crosslink the tetrafluoroethylene-propylene copolymer, peroxide crosslinking, electron beam crosslinking and the like are possible. For example, peroxide is used as a radical generator such as Peroximon F100 (manufactured by NOF CORPORATION), and crosslinking is performed using, for example, triallyl isocyanurate as a polyfunctional monomer.

In the present invention, a compounding agent other than the crosslinking agent may be added,
For example, MT carbon can be added as the carbon black, and sodium thraphosphate can be added as the release agent. The radical generator used in the present invention,
Polyfunctional monomers, carbon black, release agents, etc.
There is no particular limitation as long as it is not toxic to cells or microorganisms.

The rubber part of the present invention is not particularly limited in its type as long as it is a part in contact with the medium, for example, a rubber tube for liquid feeding,
There are rubber tubes for gas feeding, O-rings, packing, rubber sheets, rubber stoppers, and the like.

In the rubber part of the present invention, various cells or microorganisms can be cultured by a culture device using the rubber part. The cells or microorganisms to be cultured are not particularly limited, but include human uterine cancer cell HeLa, Chinese hamster lung-derived established fibroblast V-79, African green monkey kidney-derived established fibroblast Vero, human fetal lung-derived normal. Diploid fibroblast Flow-20
00, human fetal lung cells MRC-5, adhesion-dependent animal cells such as chimpanzee liver fibroblasts, lymphocytes, lymphoblasts,
Suspension-proliferating animal cells such as myeloma cells such as Burkitt's lymphoma and myeloma or hybridoma cells thereof, ceriba lauren protoplasts, hanakirin protoplasts, tobacco mesophyll protoplasts, carrot protoplasts, callus of genus moss, callus of soybean, callus of purple, strawberry Shoots, tomato shoots, potato shoots (Solanum goniocalyx) shoots, pea shoots, carnation shoots, asparagus shoots, chlorella and other plant protoplasts, callus, parts of the plant Plant cells such as eggplant tissues, yeasts such as wine yeast, baker's yeast, soy sauce yeast, feed yeast, Nocardia, actinomycetes such as Streptomyces, Escherichia coli, Bacillus subtilis, lactic acid bacteria, Ascomycetes, Ascomycetes such as Aspergillus, Shiitake, Nameko, D It may be exemplified microorganisms such as Basidiomycetes such as Length.

Further, the medium used when culturing cells or microorganisms by a culture device using the rubber component of the present invention is not particularly limited, and a known medium can be used as it is, for example, culture of animal cells. Examples of the medium used for RPMI1640 medium, Eagle MEM medium, Dulbecco's anisotropic Eagle medium, Earle medium 199, Ham F12 medium, etc., in these medium, at a ratio of 5 to 10% by volume, For example, fetal bovine serum, newborn bovine serum, horse serum,
It is preferable to add human serum for use. Serum-free medium without serum, such as HB102 (Hana Biologics), RITC55-9 medium can be used.

In addition, a rinse medium is used as a medium for culturing plant cells.
Liquid Soil of Linsmaier-Skoog, Mu
Murashige-Skoog's liquid medium, MG
-5 Soil land, which is used for culturing microorganisms
As, carbon source, nitrogen source, inorganic salts, vitamins, etc.
For example glucose, fructose, maltose, sucrose
Glucose, starch, dextrin, molasses, citric acid, fu
Malic acid, ammonium sulfate, ammonium chloride, ammonium phosphate
Gum, sodium sulfate, urea, amino acids, peptone, large
Bean whey, yeast extract, malt extract, meat extract, corn
Stapler, magnesium salt, phosphate, vitamin
B₁, Vitamin B₆, Vitamin C, etc.
Prepared by dissolving in water and agar medium etc.
To be

e. Examples Next, the present invention will be described more specifically by the following examples and comparative examples.

Example 1 Tetrafluoroethylene-propylene copolymer rubber was cross-linked into a rubber sheet having a diameter of 2 cm and a thickness of 2 mm according to the compounding formulation shown in Table 1. This rubber sheet was used for a plastic dish for cell culture ( (Diameter 60 mm, Becton and Dickinson), 5 ml of culture medium consisting of minimal essential medium containing 10 V / V% fetal bovine serum and 200 V-79 cells were added to the carbon dioxide incubator. 37 ℃, 100% humidity, 5V / V%
The cells were cultured for 7 days in an air atmosphere containing carbon dioxide. After culturing, the number of cell colonies formed from living cells was counted to measure the survival rate in the culture medium containing the rubber sheet. As a result, the number of colonies was 186, and the plate efficiency was 93%.

Example 2 A tetrafluoroethylene-propylene copolymer rubber was cross-linked into a rubber sheet having a formulation shown in Table 1 (diameter: 2 cm, pressure: 2 mm. 200 Vero cells were used with this rubber sheet). After culturing for 11 days under the same conditions as in Example 1, the survival rate was measured by counting the number of cell colonies, and as a result, the number of colonies was 190, so that the plate efficiency was 9
It was 5%.

Comparative Example 1 Instead of the rubber sheet used in Example 1, vinyl fluoride-
Hexafluoropropylene copolymer (diameter 2 cm, pressure 2 m
m-) was used to culture V-79 cells in the same manner as in Example 1. The plate efficiency after culturing was 0%, and the cells did not grow at all.

Example 3 A tetrafluoroethylene-propylene copolymer rubber was prepared according to the formulation shown in Table 1 so that the inner diameter was 3 mm, the outer diameter was 5 mm, and the length was 20.
Cross-linked into a cm rubber tube.

100 ml culture medium consisting of minimal essential medium containing 10 V / V% fetal bovine serum in a 100 ml spinner flask 100 ml V-79 cells 2 × 10 ⁶ cells and microcarriers (Cytodex 3, Pharmacia Fine Chemicals) Add 3 g and cultivate for 5 days in an air atmosphere containing carbon dioxide at 37 ° C, 100% humidity and 5 V / V% carbon dioxide while rotating the stirring blade inside the spinner flask at 30 rpm. did. During culturing, the new culture solution that has passed through this rubber tube is placed in the spinner flask.
The solution was continuously fed at a rate of 100 ml / day, and at the same time, the culture solution in the spinner flask was discharged through a nylon mesh at a rate of 100 ml / day. After 5 days, the number of cells reached 1 × 10 ⁸ cells, which was 50 times that after the start of culture, and good growth was observed.

Comparative Example 2 A vinyl chloride tube (inner diameter 3 mm, outer diameter 5 mm, length 20 cm, manufactured by Norton) was used in place of the rubber tube used in Example 3, and a spinner flask was used in the same direction as in Example 3. V-79 cells were cultured. The number of cells, which was 2 × 10 ⁶ cells at the time of disclosure of the culture, decreased to 1 × 10 ⁵ cells or less after 4 days.

Comparative Example 3 Instead of the rubber tube used in Example 3, a vinyl fluoride-hexafluoropropylene copolymer (inner diameter 3 mm, outer diameter 5
mm, length 20 cm), V-79 cells were cultured in a spinner flask by the same method as in Example 3. Start of culture 2
The number of cells, which was × 10 ⁶ , decreased to 2 × 10 ⁵ or less after 5 days.

f. Effect of the invention The rubber component of the present invention is non-toxic to cells or microorganisms, and thus the viability of cells or microorganisms does not decrease during cell culture. The culture efficiency can be improved.

Claims (1)

[Claims] 1. A rubber component for cell or microorganism culture equipment, which is formed by a crosslinked product of a tetrafluoroethylene-propylene copolymer.