KR20060041863A - Medium set for culture and method of preserving medium - Google Patents

Abstract

There is a need for a container for culturing cells that enables long-term storage of a medium containing a carbonate, and preferably enables oxygen addition to the medium during culture.

A container containing a medium containing a carbonate contains at least a medium containing a carbonate, a sealed first container and a medium component other than a carbonate, and is sealed from a second container. It provides a culture medium set, characterized in that the container is formed of a non-breathable material.

Badge set, its preservation method

Description

Medium set for culture and method of preserving medium}

1 is an embodiment of the present invention.

2 is an embodiment of the present invention.

3 is an embodiment of the present invention.

{Description of the sign}

l first container

11A medium component containing at least carbonate as the medium component (medium component A)

2 second containers

21 Medium components other than the medium component containing at least carbonate (medium component B)

3 communicating closure means

31 male connector

32 female connector

The present invention relates to a culture medium set having excellent long-term storage of a carbonate-containing medium, and preferably a culture medium set having sufficient aeration to the medium during culture.

In recent years, the field of regenerative medicine has been developed for the purpose of actively using cells for biological tissues and organs which are missing functional disorders and insufficiency, and for regeneration of the functions. The technique of taking out dry cells from a mammal, culturing the cells in vitro, and then returning them to the living body is also part of this technique. The medium used for culturing such cells is prepared in each compounding amount depending on the cells to be cultured with an energy source, nutrient, coenzyme, inorganic ion, and p H buffer. If necessary, serum and cell growth factors are also added. As an energy source, glucose or the like is used to be a fuel necessary for physiological activity of cells. Nutrients are selected from the raw materials of cells such as nucleic acids and amino acids. Coenzymes are selected to enhance and stabilize intracellular enzyme activity such as vitamins and trace metals. The inorganic ions are ions existing in living bodies such as sodium ions and potassium ions, and serve to adjust the penetration pressure of the medium. In addition, the p H buffer solution serves to adjust the p H of the medium in which the above components are blended, phosphate buffer, sodium bicarbonate, N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid (HEPES). Suitable ones are used.

Conventionally, culture of mammalian adherent cells or suspension cells in vitro has been carried out in a polystyrene chalet. However, since such a container has low gas permeability, in order to sufficiently exchange oxygen or carbon dioxide necessary for cell proliferation, it is sufficient to multiply the cell after the liquid layer of the culture solution is about 3 mm and an air layer is not provided in the container. Couldn't get it. Therefore, when culturing a large amount of cells, a large amount of space was required because of the useless space.

In order to solve such a problem, the cell culture container is disclosed as the container which consists of ionomer resin which has oxygen permeability (patent document 1). In addition, a cell culture vessel formed of a second vessel made of a liquid permeable material is disclosed inside a first vessel made of a gas permeable material, and sufficient oxygen addition is provided between the outside and the first vessel. The cell culture culture container which makes it possible to culture a cell at high density in the 2nd container inside is disclosed (patent document 2, 3). Furthermore, a culture vessel excellent in strength is disclosed by forming one side of the vessel from a gas-permeable silicone-derived styrene polymer (Patent Document 4). In addition, it is formed of a composition consisting of polyolefin resins other than poly-4-methylpentene-l resin and poly-4-methylpentene-1 resin, and has excellent oxygen permeability and light permeability, and is suitable for culturing plant cells. The container is disclosed (patent document 5).

However, such cell culture vessels have sufficient air permeability to the culture medium at the time of culture, but when a medium containing carbonate is used as a buffer, the pH of the medium due to the diffusion of carbon dioxide changes and the culture medium for cell culture is used. Long term preservation was difficult.

{Patent Document 1] Japanese Patent Application Laid-Open No. 60-160881

Japanese Patent Application Laid-Open No. 3-10675

Japanese Patent Laid-Open No. 3-l0676

{Patent Document 4} Japanese Patent Laid-Open No. 11-28083

Japanese Patent Application Laid-Open No. 2001-190267

Therefore, there is a need to provide a culture medium set that enables long-term storage of a medium containing a carbonate, and preferably has sufficient aeration to the medium during culture.

The present invention has been an example of the prior art, and enables long-term storage of a medium containing carbonate, and preferably relates to a culture medium set having sufficient aeration to the medium during culture.

That is, the present invention,

(l) a culture medium set containing a first container containing at least a carbonate containing a medium component and sealed with a non-breathable material and a second container containing a medium component other than the carbonate, the second container being sealed,

(2) the culture medium set according to (l), wherein the carbon dioxide permeation coefficient of the non-breathable material is less than about l000 c 3 / m 2 · 24hr · atm,

(3) the culture medium set according to (l), wherein the water vapor transmission coefficient of the non-breathable material is less than about 1000 g / m 2 · 24hr · atm,

(4) the culture medium set according to (l), wherein the second container is made of a breathable material,

(5) the culture medium set according to (4), wherein the oxygen permeability coefficient of the breathable material is about 100 to 5000 cm 3 / m 2 · 24hr · atm,

(6) a culture medium set according to (4), wherein the carbon dioxide permeation coefficient of the breathable material is about 1000 to 20000 cm 3 / m 2 · 24hr · atm,

(7) The culture medium set described in (1), wherein the closing means connectable to the first container and the second container is provided.

(8) a culture medium set according to (1), wherein the carbonate is sodium hydrogen carbonate, and

(9) The medium preservation method characterized by storing the medium containing at least carbonate as a medium component in a non-breathable atmosphere using the culture medium set as described in (1)-(8).

(The best form to carry out invention)

The culture medium set according to the present invention is a first container containing a medium component (hereinafter also referred to as medium component A) containing at least carbonate as a medium component and a medium component other than carbonate (hereinafter also referred to as medium component B). And a first container formed of a non-breathable material. Although the shape of a 1st container and a 2nd container is not specifically limited, A bag, a bottle, etc. are mentioned, It is preferable that it is a bag-shape formed from the sheet | seat of lightweight, easy to mass-produce, and lightweight.

The first container of the culture medium set of the present invention is characterized by being a non-breathable material. The non-breathable material means a material which is less likely to permeate carbon dioxide. Specifically, the carbon dioxide permeability coefficient at a temperature of 25 ° C., a humidity of 50%, and atmospheric pressure is less than about 1000 c 3 / m 2 · 24hr · atm, preferably It is less than about 100 cm 3 / m 2 · 24hr · atm, more preferably less than about 1 cm 3 / m 2 · 24hr · atm, and the water vapor transmission coefficient is less than about l000g / m 2 · 24hr · atm, preferably about l00g / It is less than m <2> * 24hr * atm, More preferably, it is less than about 10g / m <2> * 24hr * atm. Furthermore, it is more preferable that it is industrially excellent in moldability, and to endure gamma ray sterilization. Suitable materials to select include stretched nylon, polyester, polyvinylidene chloride polyvinylidene chloride coat stretched nylon, polyvinylidene chloride coat polyester, polyvinyl chloride coat polypropylene, polyvinyl alcohol, poly (ethylene vinyl alcohol) The copolymer 1, aluminum vapor deposition polyester, a silica coat polyester, etc. are mentioned.

The medium in the present invention basically means a general purpose composed of an energy source, a nutrient, a coenzyme, an inorganic ion, a serum, and a pH buffer. Such a medium component is prepared in each compounding quantity according to the cultured cell. The energy source is a fuel necessary for physiological activity of the cell, and may include glucose and the like. Nucleic acids, amino acids and the like are added as nutrients to become a cell constituent raw material. The amino acid is added not only essential amino acids such as varin, leucine and lysine, but also non-essential amino acids. Coenzymes have a role of enhancing and stabilizing intracellular enzyme activity such as vitamins and trace metals. Inorganic ions are ions existing in living bodies, and serve to adjust the penetration pressure of the medium. As an example, sodium ion, potassium ion, magnesium ion, calcium ion, etc. are mentioned. If necessary, serum and cell growth factors are also added. In addition, the p H buffer serves to adjust the p H of the medium in which the above components are blended, phosphate buffer, carbonate (neutral), N-2 -hydroxy ethyl piperazine -N '-2 -ethane sulfonic acid ( HEPES) etc. can be mentioned.

Moreover, you may add timely compositions or natural products other than the said component to such a medium. Examples of such additives include insulin, horbor esters, hydrocortisone, and animal-derived pituitary extracts.

The medium containing the carbonate of the present invention means a medium in which carbonate is selected as one p H buffer solution of the medium component in the medium. The other media component is not particularly limited and is timely selected for the cells to be cultured.

The carbonate in this invention produces | generates carbon dioxide when melt | dissolved in a solvent, and includes all which can be advantageous as a carbonate ion. Examples of the carbonate include sodium bicarbonate, potassium bicarbonate, magnesium carbonate and the like, but sodium bicarbonate is generally used.

The medium component (medium component A) containing at least carbonate in this invention should just contain carbonate in the medium component which comprises the medium containing carbonate, and only carbonate, carbonate and coenzyme, carbonate, and inorganic ion It is not possible to combine these and the like, and the case of the medium itself containing carbonates is also included.

At this time, the medium container (medium component B) other than the medium component A is accommodated in a 2nd container. The medium component B is not particularly limited as long as it does not contain a carbonate, and it is preferable that the target medium is prepared when the medium component A accommodated in the first container and the medium component B accommodated in the second container are mixed. desirable. For example, when carbonate is contained as the medium component A in the first container, an energy source, nutrients, coenzymes, and inorganic ions are stored in the second container as the medium component B, and as the medium component A in the first container. When the carbonate and the energy source are accommodated, the second container contains nutrients, coenzymes, and inorganic ions as the medium component B.

Moreover, it is not necessary to divide into the above categories, such as an inorganic ion and a nutrient, For example, carbonate, calcium ion, glucose, glutamic acid, etc. are accommodated in a 1st container as a medium component A, and the medium component B is used as a medium component B. You may accommodate other media components in a 2nd container.

Moreover, the medium itself may be the medium used for culture. That is, the medium itself may be accommodated in a first container formed of a non-breathable material, and whatever the medium component B corresponds to, the second container may be empty.

The 1st container and the 2nd container in this invention are each sealed, It is characterized by the above-mentioned. The sealed state means that the medium components A and B accommodated in the first container and the second container, respectively, do not leak from the container and do not mix with each other.

In addition, the material of the second container of the cell culture container of the present invention is not particularly limited, but if it is a breathable material, cells can be cultured in the second container, which is preferable. The permeability coefficient of oxygen in a breathable material at a temperature of 25 ° C. is about 100 to 5000 cm 3 / m 2. 24hr * atm, Preferably it is about 1100-3000cm <3> / m <2>, 24hr * atm, More preferably, it is about 1250-2750cm <3> / m <2>. 24 hr · atm, and a carbon dioxide permeation coefficient of about l000 to 20000 cm 3 / m 2. 24 hr · atm, preferably about 3000 to 11500 cm 3 / m 2. It is 24hr * atm, More preferably, it is about 5000-9000cm <3> / m <2> * 24hr * atm. Furthermore, it is preferable that it is more industrially excellent in moldability, and it is a thing with high transparency which can tolerate gamma-ray sterilization and can observe the shape of an internal medium. Suitable materials to be selected include polyethylene, polyvinyl chloride, poly (ethylene vinyl acetate) copolymer 1, poly (ethylene-ethyl acrylate) copolymer 1, poly (ethylene-methacrylate) copolymer, and the like. .

Furthermore, it is preferable that the 1st container and the 2nd container in this invention have a means which can be opened. Openable means that it is possible to receive each of the 1st container and the 2nd container, and to take out the sealed media components A and B from a container, and to mix these media components. That is, what is necessary is just to provide the means which can be opened by artificial external force after sealing. For example, a bag formed of a polymer material that can be sheared by artificial external force is selected as a container, and opened using a tool such as scissors, a method of pre-tearing a tear easily by hand, or a method of installing a lid or a photo on the container. Etc. can be mentioned.

Furthermore, the 1st container and the 2nd container of this invention are provided with the closing means which can communicate. The closing means is one form of achieving a sealed state, and means blocking the passage of the medium, and corresponds to the sealed state. In addition, communication possible means that it can be easily communicated by human operation. Therefore, the communicable closing means includes, for example, a male connector and a female connector when the first container and the second container are separately provided, and the first and second containers are connected to each other by connecting the male connector and the female connector. And the second container are in communication. On the other hand, in the case where the first container and the second container are installed in communication with each other, there may be mentioned the installation of a holding body such as a clip, a communication piece, a medicine seal, or the like in a communicating place. By communicating in this way, the medium component accommodated in the 1st container and the 2nd container can be mixed easily, and a medium can be easily prepared in a container.

The storage method of the medium of the present invention is characterized by storing a medium component containing at least carbonate in a non-breathable atmosphere. As one embodiment, it is effective to save using the culture medium set of the present invention.

When culturing the cells, oxygen is added using a medium containing a carbonate prepared by mixing the medium component A contained in the first container of the culture medium set of the present invention and the medium component B contained in the second container. Cells can be cultured in a sufficiently supplied atmosphere. At this time, it is preferable that the amount of the medium moving from the first container to the second container is small. That is, the form which accommodates only a carbonate as a medium component in a 1st container, and accommodates a medium component other than a carbonate in a 2nd container is preferable.

In one embodiment of culturing the cells in an atmosphere in which the oxygen is sufficiently supplied, after opening the first container and the second container with scissors or the like, each medium component is developed in a chalet to prepare a medium containing carbonate. The method of culturing as it is is mentioned.

In addition, when the second container is a breathable material, the second container itself can be cultured. That is, when the medium component A is accommodated in the 1st container and the medium component B is accommodated in the 2nd container, and the closing means which can communicate with each of the 1st container and the 2nd container is provided, the closing means communicates. After that, the medium component A contained in the first container is transferred to the second container, and after the medium containing the carbonate is prepared in the second container, the cells can be cultured in the second container as it is.

1 is one embodiment of the culture medium set of the present invention. Carbonate 11 is accommodated in the 1st container 1 formed of the non-breathable material as a medium component, and medium components 21 other than carbonate are accommodated in the 2nd container 2. As shown in FIG. The weak seal part is formed as the closing means 3 which can communicate with a 1st container and a 2nd container. The chemical seal portion can communicate with each other by pressing the first container or the second container.

Furthermore, FIG. 2 is another embodiment of the culture medium set of this invention. In the first container 1 formed of a non-breathable material, a carbonate 11 is accommodated as a medium component, and a medium component other than carbonate is accommodated in the second container 2. The 1st container and the 2nd container are connected by the tube made of vinyl chloride, and the tube is provided with the communication piece as the closing means 3 which can communicate. The communication piece is capable of recommunication by folding an internal member.

Furthermore, FIG. 3 is another embodiment of the culture medium set of this invention. In the first container 1 formed of a non-breathable material, at least a carbonate 11 is accommodated as a medium component, and a medium component 22 other than a carbonate is accommodated in the second container 2. The closing means 3 which can communicate the 1st container 1 and the 2nd container 2 is provided with the male connector 31 and the female connector 32. As shown in FIG. Communication is enabled by connecting the male connector 31 and the female connector 32.

{Example}

Although an Example demonstrates this invention in detail below, this invention is not limited to such an Example.

Example 1. Preparation of the culture medium set of Figure l

In the first container, two sheets of low density polyethylene are prepared in the inner layer of low density polyethylene, the middle layer of poly (ethylene-vinyl alcohol) copolymer 1 and the outer layer, and the polyethylene-polypropylene polymer blend ( Mixing ratio 3.7) The agent-formed sheet for forming the weak seal is arranged to be buried a predetermined length. Then, a band-shaped portion passing through the overlapping portion of the three sheets including the side edges of the two sheets and the sheet for weak sealing is thermally welded to produce a bag-shaped container having a bend. 30 ml of sodium bicarbonate aqueous solution is accommodated in this bag-shaped container, and the part of a bag-shaped container is heat-welded and produced.

In addition, after producing a tube-like film of linear low density polyethylene by the inflation method, the sheet for forming a weak seal protrudes a predetermined length in the other bent portion, and the three sheets including the sheet for weak sealing overlap each other. A band-shaped portion passing through is welded to produce a bag-shaped container having a bend. In this bag-shaped container, all the media components other than the aqueous sodium hydrogen carbonate solution are accommodated, and the bent portion of the bag-shaped container is thermally welded to prepare a second container (capacity 100 ml) 2.

Next, the first container and the second container are connected. At this time, a part of the linear low-density polyethylene part including the medicine seal forming seal which protrudes in the second container and the medicine seal part formed in the manufacturing process of the second container is also inserted into the non-heat welded part of the first container. Next, about the location (part where three sheets overlap) and the manufacturing process of the 2nd container where the sheet | seat for chemical seal formation which protruded with respect to the 2nd container, and the non-heat-welded part of the 1st container overlapped. A part of linear low-density polyethylene including the formed weak seal part and the non-heat-welded part of the 1st container are welded and connected to the strip | belt-shaped part which passes through the location (part where the five sheets overlap), and is shown in FIG. Prepare a culture medium set.

Example 2. Preparation of the culture medium set of Figure 2

The inner layer is made of low density polyethylene, the middle layer is made of poly (ethylene-vinyl alcohol) copolymer, and the outer layer is prepared with two sheets of low density polyethylene, and the two sheets are stacked so that the medium can be accommodated and a tube can be connected. In one place, the side edge of the sheet was heat welded to create a first container l. Furthermore, 30 ml of sodium hydrogencarbonate aqueous solution was accommodated from the said photo.

Furthermore, after making a tube-shaped film by the inflation method on linear low density polyethylene, the medium can be accommodated in the other part of the bend, and the photo which can connect a tube is arrange | positioned, and both the bends are heat-welded, and the 2nd The container l was produced. Furthermore, medium components other than the aqueous sodium hydrogen carbonate solution were accommodated from the photo.

Next, the first container and the second container are connected. The polyvinyl chloride tube provided with the communication piece was connected to each photo provided in the 1st container and the 2nd container, and the culture medium set shown in FIG. 2 was produced.

The culture medium set of the present invention enables long-term storage of a medium containing a carbonate and cultivation under a sufficiently aerated atmosphere.

According to the culture medium set of the present invention, since the diffusion of carbon dioxide in the carbonate-containing medium is suppressed and the pH change of the medium is suppressed, long-term storage is possible. In addition, it is possible to culture the cells in a ventilated atmosphere.

Claims (9)

A culture medium set containing a first container containing at least a carbonate containing a medium component and sealed with a non-breathable material and a second container sealed containing a medium component other than the carbonate. The culture medium set according to claim 1, wherein the carbon dioxide permeation coefficient of the non-breathable material is less than about 1000 cm 3 / m 2 · 24hr · atm. The culture medium set of claim 1 wherein the water vapor transmission coefficient of the non-breathable material is less than about l000 g / m 2 · 24hr · atm. The culture medium set according to claim 1, wherein the second container is made of a breathable material. The oxygen permeation coefficient of the breathable material is about l00 to 5000 cm 3 / m 2. A culture medium set that is 24hr · atm. The carbon dioxide permeation coefficient of the breathable material is about 1000 to 20000 cm 3 / m 2. A culture medium set that is 24hr · atm. The culture medium set of Claim 1 provided with the closing means communicable to a 1st container and a 2nd container. The culture medium set according to claim 1, wherein the carbonate is sodium bicarbonate. A medium preservation method, wherein the medium containing at least carbonate as a medium component is stored under a non-breathable atmosphere using the culture medium set according to claims 1 to 8.

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