JP2005323588A - Culturing double vessel and culturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culturing vessel needing no plurality of incubators even in case of simultaneously culturing several kinds of cells liking different gas concentrations, and to provide a culturing method using such a vessel. <P>SOLUTION: The culturing double vessel 1 comprises a gas-permeable culturing vessel 2 having an injection/ejection port 6, a gas-barrier vessel 3 covering the culturing vessel 2, and joints 12 and 13 for holding the culturing vessel 2 in the gas-barrier vessel 3 through ensuring a space to be formed between the culturing vessel 2 and the gas-barrier vessel 3. In culturing cells or microorganisms, the space formed between the culturing vessel 2 and the gas-barrier vessel 3 is accommodated with a gas with its composition controlled. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a culture vessel and culture method for cells or microorganisms, and in particular, it is excellent in storage of a culture solution, achievement of an optimal culture environment, and easy extraction of contents, and use of a carrier such as suspension cell culture or beads or nonwoven fabric. The present invention relates to a culture vessel suitable for culturing adherent cells and a culture method using the culture vessel.

Conventionally, as a culture vessel for cell culture, a medium-filled bag in which a culture solution is filled in a bag made of plastic having good gas permeability is known. For example, in Patent Document 1, a solvent injection tube in which a sterilization filter is disposed in the middle of the upper end of a bag-shaped main body made of a flexible plastic sheet and a medium outlet are mounted, and the bag-shaped main body is sterilized. A medium-filled bag in which a medium is enclosed is disclosed. In Patent Document 2, a hermetic sealed tissue culture container containing a tissue culture medium is packaged and sealed with a non-breathable material, and stored after removing the non-breathable material during tissue culture. A container for cum tissue culture is disclosed. In Patent Document 3, a culture bag made of a film having a large gas permeability and sterilized with γ rays is aseptically filled with a liquid medium subjected to filtration sterilization, and the filling port is aseptically sealed, A method for producing a culture bag containing a liquid field medium in which a secondary packaging material having a low gas permeability and sterilized by γ rays is packaged aseptically is disclosed.
Japanese Utility Model Publication No. 2-93999 Japanese Utility Model Publication No. 3-172169 Japanese Patent Laid-Open No. 4-71482

  Since cell culture is usually performed in an incubator with a controlled gas composition, when culturing multiple types of cells that prefer different gas concentrations at the same time, conventional culture bags require multiple incubators with different gas concentrations. In addition, a large space is required and the operation is complicated and time-consuming. Further, when culturing, conventional culture bags are arranged in a flat state on a net shelf in an incubator and cannot be stacked or suspended, so a large space is required.

  In addition, the conventional culture bag is thin and lacks shape stability, and is likely to be broken during the distribution process. Even when a non-breathable packaging material is used on the outside during storage as in Patent Documents 2 and 3 above, the culture bag is in a state of floating inside and is unstable in shape or is in close contact with the outer packaging material However, since it is in a state that is not much different from a single culture bag, there is no change in shape stability and there is a risk of breakage in the distribution process.

  Furthermore, the conventional culture bag is time-consuming and inefficient because the contents are taken out by gravity by hanging the culture bag when the contents are taken out after the cell culture is completed. Even if an internal pressure is applied to the culture bag to take it out, it is difficult to swell the bag itself.

  The present invention has been made in view of the above-mentioned problems in conventional culture vessels, and does not require a plurality of different incubators even when cultivating a plurality of types of cells that prefer different gas concentrations at the same time. It is possible to reduce the space, and it is excellent as a distribution container without being damaged in the distribution process, and the contents can be efficiently taken out after the cell culture is completed. The object is to provide a culture vessel.

  Another object of the present invention is to provide a method for culturing cells and microorganisms using this culture vessel.

  The culture double container according to claim 1, which achieves the above object, comprises a gas permeable culture container having an injection / outlet, a gas barrier container covering the culture container, the culture container and the gas barrier container And a culture container holding means for holding the culture container in the gas barrier container so that a space can be formed between the culture container and the gas barrier container when the cells or microorganisms are cultured. A gas whose gas composition is controlled is accommodated in a space formed between the two.

  The invention according to claim 2 is a culture double container in which the culture container holding means is means for partially joining the culture container to the gas barrier container.

  The invention described in claim 3 is a culture double container in which a gas communication port communicating with a space between the culture container and the gas barrier container is provided in the gas barrier container.

  The invention according to claim 4 is a culture double container in which the container wall of the culture container is formed into a substantially cylindrical shape, a square tube shape with a regular polygonal cross section, a spherical shape, or a hemispherical shape during culture. It is.

  According to the invention of claim 5, the culture vessel is filled and sealed with a culture solution, and the space between the culture vessel and the gas barrier container is replaced with an inert gas or a mixed gas of an inert gas and carbon dioxide gas. This is a culture double container.

  The invention according to claim 6 is a culture double container in which the culture container is filled and sealed with a culture solution and the space between the culture container and the gas barrier container is deaerated.

  The invention according to claim 7 is a culture double container in which a culture solution is filled and sealed in the culture container, and an oxygen absorbent is sealed in a space between the culture container and the gas barrier container.

  The invention according to claim 8 is a culture double container in which the gas barrier container has an oxygen absorbing part or an oxygen absorbing layer.

  According to the ninth aspect of the present invention, there is provided a gas permeable culture container having an inlet / outlet and a gas barrier container covering the culture container between the culture container and the gas barrier container. This is a method for culturing cells or microorganisms in which the gas composition is controlled in the space of the cells and the cells are cultured.

  A tenth aspect of the present invention is a culture method in which a gas whose gas composition is controlled is injected and poured out from a gas passage communicating with a space between the culture container and the gas barrier container.

  The invention described in claim 11 is a culture method for extracting the contents of the culture container by injecting a compressed gas into the space between the culture container and the gas barrier container in the culture process.

  According to the present invention, the gas having a controlled gas composition is accommodated in the space formed between the culture container and the gas barrier container during cell culture, so that a plurality of types of cells that prefer different gas concentrations are cultured simultaneously. In some cases, multiple incubators are required by placing multiple culture double containers to cultivate these cells in a common temperature-controlled room and injecting gas of the required gas concentration into these double culture containers. In addition, cell culture can be performed easily. Further, according to the present invention, since the culture container is held in the gas barrier container by the culture container holding means so that a space can be formed between the culture container and the gas barrier container, Even if the culture double containers are stacked, the gas can permeate each culture container and does not interfere with the culture. In addition, since the shape of the container does not change even if the culture container is hung vertically, it can be placed in a constant temperature room with many culture containers hung vertically, reducing the installation space compared to conventional culture containers. Can be made.

  Further, according to the present invention, by injecting a compressed gas such as compressed air into the space between the culture container and the gas barrier container after the culture, the contents in the culture container can be quickly taken out. The contents can be taken out more efficiently than by the gravity method.

  Furthermore, according to one aspect of the present invention, the culture vessel is formed in any one of a cylindrical shape, a square tube shape having a regular polygonal cross section, a spherical shape, and a hemispherical shape at the time of cultivation. Since the gas diffuses in the container from the direction of approximately 360 degrees toward the center of the container, the vicinity of the container wall and the container wall are compared with the conventional culture container composed of a flat bag. The difference in the concentration of each dissolved gas at the part farthest from the center becomes small. Therefore, the culture conditions can be made closer to the optimum culture conditions, and the culture efficiency of the cells and the cell culture activity can be increased.

  Furthermore, according to another aspect of the present invention, the space between the culture vessel and the gas barrier vessel is replaced with an inert gas or a mixed gas of inert gas and carbon dioxide, or the space is degassed. This makes it possible to more completely prevent the culture medium from being oxidized, and to extend the storage period of the culture liquid. Similarly, the same effect can be obtained by enclosing an oxygen absorbent in this space or by providing an oxygen absorbing portion or an oxygen absorbing layer in a gas barrier container.

Embodiments of the present invention will be described below with reference to the accompanying drawings.
1 to 4 show one embodiment of a culture double container according to the present invention, FIG. 1 is a plan view thereof, FIG. 2 is a view taken along arrow AA in FIG. 1, and FIG. -B perspective view, FIG. 4 is a plan view of the culture vessel.

The culture double container 1 includes a culture container 2 and a gas barrier container 3.
In the present invention, it is preferable that the culture vessel 2 is formed in one of a substantially cylindrical shape, a square tube shape having a regular polygonal cross section, a spherical shape, and a hemispherical shape during culture. In this specification, “substantially cylindrical” means not only a perfect circular cylinder as shown in FIG. 7 (a) but also a slightly modified circular cross section or ellipse as shown in FIG. 7 (d). It is meant to include a cylinder including a cross-sectional shape obtained by deforming a perfect circle to such an extent that it does not hinder the object of the present invention, such as a cross-sectional shape. Similarly, “substantially a square polygonal cross section” means that the cross section is a regular triangle, square (shown in FIG. 7 (e)), regular pentagon, regular hexagon (FIG. 7 (b)). In addition to regular polygonal square tubes such as regular octagons (shown in FIG. 7 (c)), these regular polygons are slightly modified to the extent that they do not hinder the achievement of the object of the present invention. It is meant that the cross-sectional shape thus formed also includes a square tube.

The culture vessel 2 is made of a plastic having excellent gas permeability, and includes a cylindrical portion 4 in which a plurality of (5 in the illustrated example) cylinders 5 are arranged in a beach mat shape, and both ends of the cylindrical portion 4 in the longitudinal direction. As a whole, it is a bag-like container having a rectangular shape in plan view, provided with continuously formed end portions 2a, 2b, and outer end edges of both end portions 2a, 2b are closed. The end portions 2a and 2b have a flat surface shape, and the cylinders 5 communicate with each other via the end portions 2a and 2b. The end 2 a is provided with an injection / extraction port 6 for various chemicals such as a culture solution, cells, and cell growth factors, and communicates with the inside of the culture vessel 2.
Note that the required number of injection / extraction ports 6 can be provided in accordance with the purpose, and a connection tube or a connector or the like can be provided at the tip of the connection tube as needed.

  When the culture vessel 2 is manufactured, as shown in FIG. 9, rectangular flexible plastic sheets 7 and 8 are arranged so as to face each other with an interval therebetween, and end edges 7a on four sides, 8a, the sheets 7, 8 are partially heat-sealed along the corresponding longitudinal lines 7b and 8b, 7c and 8c, 7d and 8d, and 7e and 8e of the opposing sheets 7 and 8 after being joined by heat sealing. By doing so, the cylindrical part 4 which consists of the five cylinders 5 can be formed.

  By thus heat-sealing the opposing wall surfaces of the flexible culture vessel 2 having flexibility and filling the culture solution more than a certain amount, the culture vessel 2 can be given shape retention, and the culture vessel 2 and gas barrier properties can be provided. Adhesion with the vessel 3 can be prevented or reduced, and loss of gas diffusion into the culture vessel 2 can be prevented or reduced. As a means for imparting shape retainability, in addition to heat sealing, container pressing means such as jigs, clips or magnets are used to press the flexible container walls facing each other so as to be in partial contact. be able to. Further, as means for preventing or reducing the adhesion, a member for preventing adhesion between the culture vessel 2 and the gas barrier container 3 is sandwiched, or an uneven portion is provided on each vessel wall. Also good.

As the gas permeable culture vessel 2, it is preferable to use a material having good gas permeability and capable of withstanding γ-ray sterilization. For example, polyethylene (PE), polyvinyl chloride (PVC), ethylene vinyl acetate copolymer (EVA), ethylene ethyl acrylate copolymer (EEA), ethylene methyl methacrylate copolymer (EEMA), styrene-ethylene-butylene-styrene (SEBS) A container such as a film bag or a blow bag made of a soft plastic such as polypropylene (PP) or a polymer blend with polyethylene. In addition, when sterilization is not based on γ rays, Teflon (registered trademark),
Silicone rubber, 4-methyl-1-pentene homopolymer or copolymer, and butadiene homopolymer or copolymer can be used. As the other monomer of the copolymer, for example, α-olefin having 2 to 24 carbon atoms, styrene or the like is used, and a polymer composed of one or more of the α-olefins is blended and used. You can also

  In the embodiment of FIG. 1, as shown in FIG. 3, the gas diffuses from the container wall of each cylinder 5 toward the center of each cylinder 5 from the direction of arrow C, that is, from the direction of 360 degrees. The difference in concentration of each dissolved gas at the center farthest from the vessel wall is reduced, and the culture conditions can be made closer to the optimum culture conditions. When culturing, the culture vessel 2 may be shaken, rocked or rotated at a level that does not damage the cells, if necessary, and this is observed when culturing floating cells or microorganisms. Since the sedimentation of cells or microorganisms can be prevented and the agitation of the culture solution can be promoted, it is desirable from the viewpoint of maintaining uniformity.

  As shown in FIG. 4, two sheets are heated on the outside of the culture vessel 2 in the longitudinal direction of the culture vessel 2 in the end portions 2a and 2b and on the outside of the cylindrical portion 4 in the width direction of the culture vessel. Seal portions 2c, 2d, 2e, and 2f are formed by sealing. The middle seal portion 2d of these seal portions is formed in a shape in which a portion of the gas outlet is cut out so as not to prevent the installation of a gas outlet of the gas barrier container 3 to be described later.

The gas barrier container 3 is a container having a size sufficient to allow a gas to flow between the inner culture container 2 and is formed in a rectangular shape in plan view as a whole, and its four sides are closed. It has been. The gas barrier container 3 is manufactured by, for example, superposing two gas barrier plastic sheets, thermoforming them so that the center part swells and seal parts 3a, 3b, 3c, 3d are formed on four sides. can do.
A gas inlet 9 is provided at one end of the gas barrier container 3, and a gas outlet 10 is provided at the other end, and communicates with the inside of the container 3. The gas inlet 9 and the gas outlet 10 constitute the gas inlet of the present invention. In addition, the gas inlet may serve as both the inlet 9 and the outlet 10 and is provided with at least one, preferably two or more. The composition of the culture gas may be appropriately selected according to the cells or microorganisms to be cultured. Furthermore, the culture gas can flow intermittently or continuously, and by adjusting the pressure of the culture gas, it is possible to perform culture in a state where pressure is applied or culture in a state where the pressure is changed.

  The seal portions 3a and 3b of the gas barrier container 3 are fused to the seal portions 2c and 2d so as to sandwich the seal portions 2c and 2d of the culture container 2, respectively. Joint portions 12 and 13 of the container 2 are formed. The junctions 12 and 13 have a function of holding the culture vessel 2 in the gas barrier container 3 so that a space can be formed between the culture vessel 2 and the gas barrier vessel 3. A culture container holding means is comprised.

  As shown in FIG. 5, the gas inlet can be formed in advance at the end of the gas barrier container 3 during the production of the culture double container, or the culture double container in which no gas inlet is provided. As shown in FIG. 6, the gas passage port 14 can be attached to a portion other than the seal portion of the gas barrier container 3 as a retrofit as shown in FIG.

  The gas barrier container 3 may be a single layer or multilayer blow bag or a single layer or multilayer film bag, and is not particularly limited. In the case of a single-layer blow bag or film bag, a film of a material that is easy to heat seal with the surface layer of the culture vessel and has flexibility, such as polyethylene (PE), polypropylene (PP), ethylene-olefin copolymer A plastic film made of ethylene-vinyl acetate copolymer (EVA) or the like is preferable. In particular, it is preferably made of the same or the same material as the surface area of the container body, and is preferably used in combination with an oxygen absorbent.

  In the case of imparting high barrier properties, it is preferable to use a multilayer film, and as the material having gas barrier properties, for example, biaxially stretched polyethylene terephthalate film, biaxially stretched nylon film, or other single layer or multilayer stretched film, Synthetic resin films such as ethylene-vinyl alcohol copolymer (EVOH), polyglycolic acid, aromatic polyamide, polyvinylidene chloride (PVDC), various polyvinylidene chloride coated films, alumina-deposited or silica-deposited polyester, polyamide films, etc. A multilayer film using the plastic film as a sealant is preferably used.

  In addition to coextrusion blow molding, any known lamination method such as dry lamination, sandwich lamination, extrusion lamination, etc. can be applied to the multilayering.

  In the distribution stage of the double culture vessel 1, the space between the culture vessel 2 and the gas barrier vessel 3 is replaced with an inert gas or a mixed gas of inert gas and carbon dioxide gas, or this space is deaerated. By doing so, the preservation | save property of a culture solution can be improved and a preservation | save period can be extended. Similarly, the same effect can be obtained by enclosing an oxygen absorbent in this space or by providing an oxygen absorbing portion or an oxygen absorbing layer in the gas barrier container 3.

  Any known oxygen absorber or oxygen absorbing resin can be used. For example, the iron-based oxygen absorber is preferably a mixture of iron powder and metal halide made of iron or an iron-based compound. The iron powder is not particularly limited as long as it causes an oxygen absorption reaction, for example, iron powder such as reduced iron powder, sprayed iron powder, electrolytic iron powder, pulverized products of various iron such as cast iron and steel materials, Grinded products and iron-based compounds such as iron carbide, iron carbonyl, ferrous oxide, ferrous hydroxide, and iron silicate are used. In general, the particle size is preferable, and the particle size is preferably in the range of 1 to 80 μm in consideration of handling properties, the thickness of the oxygen absorbing layer can be reduced, and the unevenness of the oxygen absorbent appearing in the film appearance. In particular, the range of 1 to 50 μm is preferable.

  Examples of the metal halide that is an auxiliary agent for the iron-based oxygen absorber include alkali metal or alkaline earth metal chlorides, bromides, or iodides, such as lithium, sodium, potassium, magnesium, calcium, and barium. Chloride or iodide is preferably used. The blending amount of the metal halide per 100 parts by weight of the iron powder is preferably 0.1 to 20 parts by weight, particularly preferably 0.1 to 5 parts by weight. It is preferable to add the metal halide in advance by mixing with iron powder because they are not easily separated. A preferable oxygen absorbent is an iron powder-based composition containing iron powder and a metal halide, and particularly preferably a metal halide-coated iron powder-based composition in which a metal halide is attached to the iron powder.

  The iron-based oxygen absorbent is packed in a space formed between the gas permeable culture container 2 and the gas barrier container 3 covering the gas permeable culture container 2 or a space connected thereto so as not to leak into the nonwoven fabric, paper or the like. It may be used, or may be mixed with some resins such as spouts and caps constituting the container. In addition, it can be used as an oxygen absorbing layer in any layer of the gas barrier container 3, so that it can be observed on one side or a part of the gas barrier container 3 so that the inside of the gas barrier container 3 can be observed. Is preferred.

The blending amount of the iron-based oxygen absorbent with respect to the resin is preferably in the range of 10 to 70% by weight, and more preferably in the range of 10 to 60% by weight. When the blending amount of the oxygen absorbent is lower than 10% by weight, the oxygen absorbing ability is insufficient, and when it is higher than 70% by weight, it may be difficult to process by injection molding, compression molding, film forming process or the like. is there. The film thickness of the oxygen absorbing layer is preferably in the range of 10 to 100 μm, particularly preferably in the range of 20 to 80 μm, regardless of the constituent materials.
When the film thickness of the oxygen absorbing layer is thinner than 10 μm, the amount of oxygen absorbent per unit film area decreases, and sufficient oxygen absorbing performance may not be obtained. On the other hand, if it is thicker than 100 μm, the total thickness of the film becomes thick, which may cause inconvenience in handling and a problem in cost.

  As the oxygen-absorbing resin, those containing a metal-based oxidation catalyst and an oxidizing resin or an oxidizing organic component, and those containing a polyphenol, ascorbic acid or the like and a basic substance are used.

  The oxidizing resin or the oxidizing organic component is a resin or an organic component that is oxidized by oxygen in the air by the action of the transition metal catalyst.

  The oxidizing resin includes (1) a resin having a tertiary carbon such as polypropylene, (2) a resin having a carbonyl group such as an ethylene-carbon monoxide copolymer, and (3) a polyamide resin having a benzene ring such as MXD6. (4) Resins having an unsaturated double bond in the main chain, such as polybutadiene, polyisoprene and copolymers thereof, (5) Resins having an unsaturated double bond such as a cyclohexene group in the side chain (6) Cyclic conjugated diene resins such as polycyclohexadiene are used. Examples of the oxidizing organic component include (7) ascorbic acid, (8) cysteine, and the like, which absorb moisture and absorb oxygen in the presence of a basic substance such as sodium carbonate and potassium acetate.

  The metal-based oxidation catalyst is preferably a group VIII metal compound of the periodic table such as iron, cobalt, nickel, etc., but also copper, silver, etc. (Group I), tin, titanium, zirconium, etc. (Group IV) ), Vanadium (Group V), chromium and the like (Group VI), manganese and the like (Group VII). Among these metal compounds, a cobalt compound is particularly suitable because of its high oxygen absorption rate. The transition metal catalyst is generally used in the form of a low-valent inorganic acid salt, organic acid salt or complex salt of the transition metal. These catalysts are preferably used in an amount of 100 to 2000 ppm per resin.

  The oxidizing resin or the oxidizing organic component is preferably applicable without impairing the transparency with respect to the case where the iron-based oxygen absorbent is used. One or both of the gas-permeable culture container 2 and the gas-barrier container 3 are preferably used. It can be used as an oxygen absorbing layer on any part or the entire surface. Further, it may be used by mixing with a part of resin such as a spout, a cap or the like, which is located in a space formed between the culture vessel 2 and the gas barrier vessel 3 or a space connected thereto. it can.

As a material configuration example in the case where an oxygen absorption layer is provided in the gas barrier container 3, it is not limited to this,
Oxygen absorbing layer (single layer)
Outer layer / oxygen absorbing layer (in the case of two layers)
Outer layer / oxygen absorbing layer / sealant layer Outer layer / gas barrier layer / oxygen absorbing layer / sealant layer Outer layer / gas barrier layer / oxygen absorbing layer / gas barrier layer / sealant layer, etc.

  When producing the culture double container of this embodiment, after producing a culture double container in which an oxygen absorbent is sealed, if necessary, sterilized with γ rays (or UV sterilization, electron beam sterilization, heat sterilization), and then The culture solution is aseptically filled, and the filling port is aseptically sealed to complete the production.

  As shown in Fig. 8 (a), when the conventional container is hung during culture, the shape is deformed and the non-uniformity of gas diffusion near the container wall and the part farthest from the container wall becomes severe. Since the gas composition cannot be achieved, it cannot be suspended in this way. However, the culture double container of the above embodiment can be shaped even if the culture double container is suspended vertically during culture as shown in FIG. 8 (b). Therefore, a large number of culture double containers 1 can be arranged in a vertically suspended state, and the arrangement space can be reduced.

  10 and 11 show another embodiment of the culture double container according to the present invention. In this embodiment, after manufacturing the culture container 2 provided with the injection / extraction ports 6a and 6b, two barrier materials are produced. As shown in FIG. 11, the culture container 2 is heat-sealed so as to cover the barrier container 3, and the culture double container 1 is manufactured.

  In this example, first, the injection / extraction port / gas discharge port storage unit 16, the gas injection port storage unit 17, and the injection / extraction port storage unit 18 are provided. The portion 19 partitioned by C is used as an unsealed portion, and the injection / extraction ports 6 a and 6 b, the gas injection port 9, and the gas discharge port 10 are covered with a barrier material constituting the gas barrier container 3. At this time, an oxygen absorbent may be enclosed in the injection / extraction port / gas discharge port storage unit 16. By doing in this way, while being able to provide the function which removes the oxygen in a container main body via the gas exhaust port 10, an oxygen absorbent can also be removed at the time of use.

Next, one or a plurality of the culture double containers 1 manufactured in this way are put in a sterilization bag and subjected to γ-ray sterilization. Thereafter, the container is unpacked in an aseptic environment, and after aseptically filling the culture solution from the injection / extraction port 6a in the unsealed portion 19 opened along C-C, it is aseptically sealed. Then, heat sealing is carried out leaving the inlet accommodating part 18, and it is set as a culture | cultivation double container.
By doing in this way, the advantage that the injection | pouring / extraction port 6a, 6b is protected by a barrier material is acquired.

  At the time of use, the extraction port / gas discharge port storage unit 16 is opened along the line DD, but the injection / extraction port 6b is already in a sterilized state by γ-ray sterilization, so that cells and the like are injected. In doing so, there is an advantage that only the outside is disinfected and the extraction port 6b need not be sterilized.

  Furthermore, when the gas discharge port 10 is also used as the gas injection port 9, the gas discharge injection port 9 and the gas injection port storage portion 17 do not have to be provided, and even if provided, they need not be opened.

  In the above-described embodiment, the culture container holding means is constituted by the joining portions 12 and 13. However, the culture container holding means is not limited to such a joining means, for example, as shown in a sectional view of FIG. Cultivation is performed by forming culture vessel support arms 15 in the longitudinal direction of the vessel in contact with the four outer corners of the culture vessel 2 at the four inner corners of the gas barrier vessel 3 made of rigid plastic. The container 2 may be held. In short, the culture container 2 may be held in the gas barrier container 3 so that a space can be formed between the culture container 2 and the gas barrier container 3. Any means can be used as long as it is possible.

  In the above embodiment, the culture vessel is configured by arranging a plurality of cylinders in parallel in a beach mat shape, but the culture vessel may be formed of a single cylinder. The same applies to the case where the container wall is formed in a rectangular tube shape, a spherical shape, or a hemispherical shape, and various modifications can be considered.

  The culture container and the gas barrier container may each be a pouch made of a flexible plastic, may be a container such as a rigid bottle, or a combination of these. May be.

13-17 is sectional drawing in the same cross section as FIG. 2 which shows other embodiment of this invention. In the example of FIG. 3, the culture vessel 2 is heat-sealed on the opposing wall surface, has a beach mat shape, and is given shape retention, but FIGS. The case where seal is not given and shape retainability is not given is illustrated. That is, in the present invention, a space can be formed between the culture container 2 and the gas barrier container 3 to prevent or reduce the loss of gas diffusion into the culture container 2, Any means may be employed as long as it can hold the culture vessel 2 in the gas barrier container 3, and FIGS. 13 to 17 show examples thereof and will be described below. 13 to 17, the same components as those of the embodiment of FIGS. 1 to 12 are denoted by the same reference numerals, and the description thereof is omitted.
In the embodiment of FIG. 13, the gas barrier container 3 is a rigid plastic container, and the gas permeable culture container 2 is a flexible flexible plastic container, Between 3, a breathable mat 17 made of a breathable material such as a nonwoven fabric is spread. A space for accommodating a gas whose gas composition is controlled is formed between the containers 2 and 3 by the breathable mat 17. In the cell culturing process, cell proliferation may be observed with a microscope or the like. Therefore, the air-permeable mat 17 does not necessarily have to be provided on the entire surface, and a part of the air-permeable mat 17 may be missing. That's fine. Further, the gas-permeable container 3 may be bonded or fixed by a known means so that the air-permeable mat 17 is not displaced.
In this embodiment, since the inner end 6a of the injection / extraction port 6 for the culture solution or the like does not protrude into the culture vessel 2, a part of the culture solution is placed in the culture vessel 2 when the culture solution is taken out. It is convenient because it does not remain.

  In the embodiment of FIG. 14, both the gas barrier container 3 and the culture container 2 are containers made of flexible plastic, and a breathable mat 17 is laid between the containers 2 and 3. Thus, a space for accommodating gas is formed. When the double container 1 is suspended in the vertical direction, both sides of the container are supported by a reinforcing jig 18 made of a rigid member such as a plate or a plurality of rod-shaped members, and the same as in FIG. Even if the culture container 2 does not have a beach mat shape, it is possible to obtain a shape retaining property.

  In the embodiment shown in FIG. 15 (b) which is a cross-sectional view of FIG. 15 (a) and an EE cross-sectional oblique view (shown by removing the gas barrier container 3) of FIG. 15 (a), the gas barrier container 3 and both of the culture containers 2 are containers made of flexible plastic, and a rectangular frame shape is formed between the inner flat surface periphery of the container 3 and the outer flat surface periphery of the container 2, respectively. A reinforcing jig 19 composed of a rigid member and a net 20 stretched on the frame-like member is interposed, and the reinforcing jig 19 forms a gas storage space between the containers 2 and 3. Has been. The inner side of the gas barrier container 3 and the reinforcing jig 19 are not fixed so that a gap 21 is formed between the inner side of the gas barrier container 3 and the outer side of the reinforcing jig 19. When the contents in the culture vessel are taken out by injecting a compressed gas such as compressed air into the space between the three, the flow of the compressed gas is not disturbed.

  In the embodiment of FIGS. 16 and 17, the gas barrier container 3 is a container made of plastic having rigidity only on one side and the other side is made of flexible plastic, and is a gas permeable culture container. 2 is a container made of a flexible plastic, and the side of the culture container 3 facing the rigid side of the gas barrier container 3 is in close contact with the inside of the gas barrier container 3, and the opposite side. A space for accommodating gas is formed only between the surface of the gas barrier and the inner side of the flexible side of the gas barrier container 3. In these embodiments, the container walls on both sides of the gas barrier container 3 may be flexible or rigid as required.

It is a top view of a culture double container concerning one embodiment of the present invention. It is AA sectional drawing of FIG. It is a BB cross-sectional perspective view of FIG. It is a top view of a culture container. It is a perspective view which cuts and shows a part of gas barrier container in order to show a gas-attachment opening of a front. It is sectional drawing of the culture | cultivation double container which shows a retrofit gas port. It is sectional drawing which shows the various shapes of a container wall. It is a sectional side view which shows the state which suspended the conventional container and the culture | cultivation double container of this invention vertically, respectively. It is sectional drawing which shows the manufacturing method of a culture container. It is a top view of the culture container in the culture double container concerning other embodiment of this invention. It is a top view of the culture double container concerning other embodiments of the present invention. It is sectional drawing which shows the other example of a culture container holding means. It is sectional drawing which shows other embodiment of this invention. It is sectional drawing which shows other embodiment of this invention. It is a figure which shows other embodiment of this invention, (a) is sectional drawing, (b) is EE sectional perspective view of (b). It is sectional drawing which shows other embodiment of this invention. It is sectional drawing which shows other embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Culture double container 2 Culture container 3 Gas barrier container 5 Cylinders 12, 13

Claims (11)

 A gas-permeable culture container having an injection / outlet, a gas barrier container covering the culture container, and a space formed between the culture container and the gas barrier container so as to form a space; A culture container holding means for holding the container in the gas barrier container, and a gas whose gas composition is controlled in a space formed between the culture container and the gas barrier container when cells or microorganisms are cultured. A culture double container characterized by containing.  The culture double container according to claim 1, wherein the culture container holding means is means for partially joining the culture container to the gas barrier container.  The culture double container according to claim 1 or 2, wherein a gas communication port communicating with a space between the culture container and the gas barrier container is provided in the gas barrier container.  The culture according to any one of claims 1 to 3, wherein a vessel wall of the culture vessel is formed into a substantially cylindrical shape, a square tube shape having a regular polygonal cross-section, a spherical shape, or a hemispherical shape during culture. Double container.  The culture container is filled and sealed with a culture solution, and the space between the culture container and the gas barrier container is replaced with an inert gas or a mixed gas of an inert gas and a carbon dioxide gas. A culture double container according to any one of the above.  The culture double container according to any one of claims 1 to 4, wherein the culture container is filled and sealed with a culture solution, and a space between the culture container and the gas barrier container is deaerated.  The culture double container according to any one of claims 1 to 6, wherein the culture container is filled and sealed with a culture solution, and an oxygen absorbent is sealed in a space between the culture container and the gas barrier container. .  The culture double container according to any one of claims 1 to 7, wherein the gas barrier container has an oxygen absorbing part or an oxygen absorbing layer.  The gas composition is controlled in a space between the culture container and the gas barrier container of a culture double container composed of a gas permeable culture container having an injection / outlet and a gas barrier container covering the culture container. A culture method for culturing.  The culture method according to claim 9, wherein a gas having a controlled gas composition is injected and poured out from a gas passage communicating with a space between the culture container and the gas barrier container. The culture method according to claim 9 or 10, wherein the contents of the culture container are extracted by injecting a compressed gas into a space between the culture container and the gas barrier container in the culture process.

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