JPH0339087A - Lyophilized medium and bag containing same medium - Google Patents

Abstract

PURPOSE:To facilitate preparation and to improve preservation and transportation by lyophilizing a medium containing one or more amino acid components, one or more vitamin components and one or more inorganic salt components. CONSTITUTION:One or more amino acid components such as L-alanine or Lcystine, one or more vitamin components such as biotin or choline chloride, one or more inorganic salt components such as NaCl or MgSO4.7H2O and a desired amount of powder medium are dissolved in sterile water to give a liquid medium, which is put in a given container and lyophilized in vacuum to give a lyophilized medium 9. Then a bag main body 2 made of gas impermeable flexible synthetic resin such as polyester is equipped with a tube 3 to inject a solvent such as distilled water and a medium outlet 4 covered with a protector 8 to make a medium-containing bag 1. Then before the injecting tube 3 provided with a removal filter 6, a clamp 7 and a connecting needle 5 to a solvent container is coupled with the main body 2, the lyophilized medium 9 is fed from the vicinity of the connecting part to the main body 2 to produce a lyophilized medium-containing bag.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a culture medium and a bag containing the culture medium that are generally used for culturing, and are suitable for culturing cells, microorganisms, etc., whether for research or industrial production. In particular, it relates to a freeze-dried medium and a bag containing the medium.

[Conventional technology and problems with the prior art]

When culturing animal cells, media are used to provide nutrients to these cells, and these media contain amino acids,
It is prepared by mixing various reagents such as vitamins and inorganic salts, and is usually commercially available in the form of liquid or solid powder. However, these liquid media and powdered media have the following disadvantages. In the case of a powdered medium, when using it, dissolve a predetermined amount in a predetermined amount of water to make an aqueous solution, add a predetermined antibiotic to the aqueous solution, adjust the medium, and then pass it through a sterilization filter. The cells were then transferred to a culture container for use. However, since these media are difficult to dissolve in water, it takes time and effort to prepare them, and since they must be introduced into the culture container through a sterilization filter, it is troublesome to introduce them into the culture container. In the case of a liquid medium, prepare the powdered medium by dissolving it in water as described above, and then perform the prescribed sterilization process. It is stored in a tank or the like for a predetermined period of time and used by introducing the required amount into a culture container. However, liquid media have the disadvantage that when stored for a long period of time, the pH changes, making them unusable for cell culture. Also, when storing it, it is stored in a large tank, which is heavy and inconvenient to carry. In order to solve the above problems, the inventors of the present invention conducted extensive studies and succeeded in inventing a medium that is easy to adjust and can be stored for a long period of time by freeze-drying the medium. Furthermore, by enclosing the freeze-dried medium in a flexible bag, we succeeded in providing a better medium-containing container. [Means for Solving the Problems] The culture medium of the present invention contains a protein component consisting of at least one type of amino acid component, at least 61 types of vitamin components, and at least one type of inorganic salt component. This is a medium obtained by freeze-drying a serum-free medium containing the serum-free medium under vacuum. Amino acid components include L-alanine, L-arginine, L-arginine hydrochloride, L-cystine, L-cystine hydrochloride, L-cystine lactate (l-hydrochloride, mono), L-cystine sodium, L-cystine Glutamic acid, L-histidine, L-histidine hydrochloride (monohydrate), L-inleucine, L-leucine, L-lysine, L-lysine hydrochloride, L
-Methionine, L-phenylalanine. L-threonine, L-tryptophan, L-tyrosine,
Several types of sodium L-tyrosine, L-valine, L-aspartic acid, L-ascorbic acid, L-serine, and L-proline are used, and the vitamin components include biotin, choline chloride, and choline bitartrate. , folic acid, D
- Calcium pantothenate, isoinositol, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, sodium a-tocopherol phosphate, vitamin B, paraaminobenzoic acid, etc. are used, and as inorganic salt components, Potassium chloride, sodium bicarbonate, sodium chloride, anhydrous calcium chloride, anhydrous magnesium chloride. Anhydrous magnesium sulfate, anhydrous sodium dihydrogen phosphate, sodium hydrogen phosphate 7-hydrate fNaJPO44) IJ
I Sodium dihydrogen phosphate・L water (NaHtPO4・H
2O) Magnesium sulfate 7 water (MgSO, 7H, 0
At least one or more of potassium dihydrogen monophosphate, etc. is used, and ingredients other than the above include calcium butyrate, D-glucose, phenol red, sodium acetate, succinic acid, HEPES,
Albumin etc. are used. The method for producing the freeze-dried medium is as follows. In the first step, a predetermined amount of a field culture medium is dissolved in a predetermined amount of unzoned water to obtain a predetermined amount of a liquid medium. In the second step, a predetermined amount of the liquid medium is placed in a container with a predetermined capacity, and the container is placed in a freezing tank of a vacuum freeze dryer to freeze the liquid medium. In the third step, the container is connected to a vacuum freeze dryer and dried for a predetermined period of time. The freeze-dried medium obtained in the manner described above is used by being sealed in a medium-containing bag shown in FIG. The culture medium bag 1 is made of materials such as ethylene-vinyl alcohol, polyvinylidene chloride, polyamide, polyester,
Acrylonitrile (AN) and methyl acrylate (MA) in acrylonitrile loop sitadiene rubber (NBR)
A gas-impermeable flexible synthetic resin made of plasticizer-free soft polyvinyl chloride (product name: S-Medica V), (product name: Zeclon), etc., which are graft-copolymerized with It consists of a bag-like main body 2, a solvent injection tube 3 such as distilled water connected to the upper end of the main body 2, and a culture medium outlet 4 covered with a protector 8. A connecting needle 5 for connecting to a container containing a solvent such as distilled water is formed at the tip of the solvent injection tube 3, and a sterilizing filter 6 and a clamp 7 are arranged in the middle. The material of the sterilizing filter can be polyvinylidene 2-fluoride, polytetrafluoroethylene, cellulose ester, etc., and a filter with a pore size of 0.22μ can prevent the invasion of bacteria. Furthermore, the above-mentioned freeze-dried medium 9 is sealed inside the main body 2. The freeze-dried culture medium 9 may be sealed inside the main body 2 by sealing the bag-like main body 2 into a bag shape with a sheet or an extruded tube, or by sealing the freeze-dried culture medium 9 inside the main body 2. After forming, before connecting the oil medium injection tube 3 to the bag-like main body 2, insert the main body 2 from the vicinity of the connection part.
A culture medium may be introduced into the container. Alternatively, the freeze-dried medium may be dissolved in a predetermined amount of solvent to form a liquid medium, and this may be sealed in a medium bag. Before use, these medium-containing bags 1 are subjected to radiation sterilization, EOG sterilization, etc. to make them sterile. [Example] A method for producing Aitane Kaiyu freeze-dried medium will be described below. 10.4 g of powdered RPMI 1640 medium manufactured by Sigma and 2 g of sodium bicarbonate were dissolved in plain water over 10 minutes to prepare a 1β liquid medium. Next, 100 m12 of the liquid medium was added for 500 m! Place the container in a vacuum freeze dryer (manufactured by Tokyo Rikakiki,
The medium was placed in a freezing tank (type FD-1) and frozen for 15 minutes until the medium was frozen to the wall of the container. Furthermore, the container was connected to the vacuum line of a vacuum freeze dryer and dried for 22 hours at a vacuum degree of 76 + nmHg and a cooling temperature of -45°C. After drying, a porous solid lyophilized medium was obtained. When the same amounts of these freeze-dried media and the powdered PRMII 640 medium were collected and dissolved in the same amount of aqueous solution and the dissolution rates were visually compared, it was confirmed that the freeze-dried media dissolved more quickly in the aqueous solution. Ta. This is thought to be because the freeze-dried medium is a porous solid and thus has a larger surface area than the powdered medium. A method for adjusting a culture medium using the culture medium-containing bag of the present invention is as follows. A connecting needle 5 of a culture medium bag 1 is connected to the mouth of a plastic bottle 10 containing distilled water, and distilled water is injected into the main body 2 via the culture medium tube 3 due to the drop due to gravity. Bacteria existing in the distilled water are captured by the sterilizing filter 6 and there is no possibility of them entering the main body 2. After sealing and cutting the injection tube 3 near the upper end of the main body 2, the main body 2 is thoroughly stirred and the culture medium is dissolved in distilled water to prepare a predetermined culture medium. When using this culture medium, the protector 8 is broken to expose the culture medium outlet 4, and the attached communication needle 11 is connected to one end of the connection tube 12, and the other end of the connection tube 12 is connected to this. Connect the connecting needle 11 connected to one end to the medium inlet 14 (or 1
4a), and the medium is led out to the culture bag 13 (or concentrated culture bag 13a) through the communication tube 12 for culture. [Effects of the Invention] As explained above, (1) the freeze-dried medium of the present invention is easily dissolved in a solvent, and a liquid medium can be easily prepared. ■Medium bags containing freeze-dried media are easy to sterilize, long-term storage, and large-volume storage. It has excellent transportability. This is an excellent invention that has the following effects.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of a bag containing a culture medium, FIG. 2 is a state diagram when adjusting the bag containing a culture medium, and FIG. 3 is a schematic diagram when drawing out the culture medium from the culture medium bag. In the figure, l is a bag containing the medium, and 2 is the main body. 3 is a solvent injection tube, 4 is a medium outlet, 5 is a connecting needle,
6 is a sterilization filter, 7 is a clamp, 8 is a protector, 9 is a freeze-dried medium, 11 is a connecting needle, 12 is a connecting tube, 13 is a culture bag, 13a is a concentrated culture bag, 29
30 indicates a liquid medium in which a freeze-dried medium has been dissolved, and a medium inlet.

Claims (2)

[Claims] (1) Freeze-dried medium containing at least one or more types of amino acid components, at least one or more types of vitamin components, and at least one or more types of inorganic salt components, which is characterized by being subjected to freeze-drying treatment. Culture medium. (2) A solvent injection tube with a sterilization filter and a medium outlet are attached to the upper end of the bag-like body made of a gas-impermeable flexible synthetic resin sheet. A medium-containing bag characterized by containing a dry medium.