JP2008212049A - Culture bag and culture apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture bag and a culture apparatus capable of readily carrying out the mass-culture of microorganisms at low cost, while supplying air at high efficiency. <P>SOLUTION: The culture bag is a bottom-closed packaging bag capable of holding a liquid in a liquid-tight state and self-supportable in a state filled with a liquid, and contains an air-feeding pipe having an attached weight. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a culture bag and a culture apparatus capable of simply and efficiently culturing cells, microorganisms and the like (in the present specification, these may be collectively referred to as “microorganisms”).

Culture of microorganisms, insect cells, plant cells, animal cells and the like is widely used for the production of substances such as proteins useful as pharmaceuticals, foods and the like. However, conventional glass and stainless steel incubators are expensive, and a large sterilizer is required to sterilize the incubator, or large-scale equipment such as a sterilization function is attached to the incubator itself. It was necessary. Furthermore, in order to repeatedly use the incubator, there is a problem that it takes time for internal cleaning and maintenance of the apparatus.
In order to solve such problems, a bag-shaped culture tank has been proposed (Patent Document 1). However, this culture tank seals the open part of the bag with a lid after storing the culture medium or culture medium, and there is a possibility that various germs may be mixed when the incubator is assembled or when the culture medium or culture medium is stored. As a result, there was a limit to maintaining the sterility inside the incubator. In addition, a container for shaking culture (Patent Document 2) made of a gusset bag and a culture container (Patent Document 3) made of a self-supporting packaging bag have been proposed, but these containers also have room for improvement in the air supply efficiency. .
Japanese Patent Laid-Open No. 06-153902 JP-A-10-150972 JP 2002-101867 A

  The present invention has been made to solve the above problems, and it is an object of the present invention to provide a culture bag, a culture apparatus, and a method for handling them, which can perform microorganism culture and various operations on microorganisms inexpensively and easily. And

  As a result of intensive studies to solve the above problems, the present inventors have added a weight to a culture bag made up of a bottomed packaging bag that can store liquid in a liquid-tight manner and can stand alone when storing the liquid. It has been found that the problem can be solved by installing the supplied air supply pipe, and the present invention has been completed.

In order to solve the above-mentioned problems, the present invention is a culture bag made of a bottomed packaging bag that can store a liquid in a liquid-tight manner and can stand alone when the liquid is stored, and a weight is added to the culture bag. A culture bag with an air supply pipe is provided.
Here, it is preferable that a weight is added so that a part of the air supply pipe contacts the bottom surface of the culture bag when the culture bag is self-supported, and the air supply pipe includes a plurality of air supply holes. In addition, it is more preferable that a rod-like weight is added along the air supply pipe so that the portion having the plurality of air supply holes lies on the bottom surface.
And it is preferable that the said packaging bag is a gusset bag.
In the culture bag of the present invention, it is preferable that the packaging bag further includes a hanging portion at the top.

The present invention further provides a culture apparatus in which the culture bag is supported by a support device that supports at least the bottom surface of the culture bag. Here, it is preferable that the support device further supports an upper portion of the culture bag. Moreover, it is preferable that the support device includes a swing device that can swing in the horizontal direction.
The culture apparatus of the present invention preferably further includes a temperature sensor.
The culture apparatus of the present invention preferably further includes a heating device.

  The present invention further provides a method for culturing microorganisms, wherein the microorganisms are cultured by supplying gas from the supply pipe using the culture bag or the culture apparatus. Here, it is preferable to shake and culture the microorganism by shaking the culture bag.

The present invention further provides a medium-containing culture bag in which the medium is sealed in a culture bag and maintained in a sterile state.
The present invention further provides a method for transporting a culture, in which a culture cultured using the culture bag is transported without being removed from the culture bag.

The culture bag of the present invention is a culture bag made of a bottomed packaging bag that can store liquid in a liquid-tight manner and is capable of self-supporting when the liquid is stored, and has a supply pipe with a weight added to the culture bag. Since it is a culture bag provided, it is possible to simply carry out the culture while allowing the air supply tube to settle and supplying air efficiently. In addition, it is preferable that a weight is added so that a part of the air supply pipe comes in contact with the bottom surface of the culture bag when the culture bag is made self-supporting. Further, when the air supply pipe has a plurality of air supply holes and a rod-like weight is added along the air supply pipe so that a portion having the plurality of air supply holes lies on the bottom surface, the air supply is uniformly supplied. This is preferable.
The culture bag of the present invention can be folded and is light, so that handling such as transportation and sterilization is easy.
Moreover, it is preferable that the packaging bag is a gusset bag because the housing efficiency is high and it is self-supporting.
Moreover, when the said packaging bag is provided with a hanging part in the upper part, it can be made to support to a support member stably.

Since the culture bag of the present invention is supported by the support device that supports at least the bottom surface of the culture bag, the culture device of the present invention can be cultured with the culture bag being stably supported. Further, when the support device further supports the upper part of the culture bag, the culture can be performed more stably. Furthermore, if the support device is provided with a swinging device that can swing in the horizontal direction, shaking culture can be performed easily and inexpensively.
In addition, when the culture apparatus includes a temperature sensor, temperature management becomes possible.
Moreover, when the said culture apparatus is equipped with a heating apparatus, it will heat and culture | cultivation will be attained.

  The culture method of the present invention is a method of cultivating microorganisms by supplying gas from the air supply pipe using the culture bag of the present invention or the culture apparatus of the present invention. It can be carried out. Microorganisms can be propagated more efficiently by shaking the culture bag and culturing the microorganisms with shaking.

The culture bag containing the culture medium of the present invention is a culture bag sealed in the culture bag and maintained in a sterile state. Therefore, culture can be performed simply by inoculating microorganisms into the culture bag containing the culture medium. It can be carried out.
The method for transporting a culture of the present invention is a method of transporting a culture cultured using the culture bag of the present invention without taking it out of the culture bag, and culturing without refilling the culture into another transport container. This is preferable because the entire bag can be transported.

The packaging bag that is the main body of the culture bag of the present invention is a bottomed packaging bag that can store liquid in a liquid-tight manner and can stand alone when the liquid is stored. The shape is not particularly limited as long as it is a bottomed packaging bag that can store liquid in a liquid-tight manner and can stand alone when storing the liquid. For example, a gusset bag, a standing pouch, a rectangular cylindrical film, and a rectangular bottom surface. Examples include a square bottom bag with a film heat-welded. Of these, a gusset-shaped packaging bag is preferable because it is easy to manufacture, can stably store more medium, and can be cultured uniformly.If the lower corner is heat-sealed diagonally, the contents are stored in the corner. Is more preferable because it does not enter.

  As such a gusset bag, a commercially available gusset-type bag-in-box (BIB) inner bag, for example, an inner layer as described in JP-A-2001-253474 and JP-A-2003-191967 has a thermoadhesive synthetic resin. A gusset bag composed of a packaging material having a layer may be mentioned. Specific examples of commercially available gusset-type bag-in-box (BIB) inner bags include Z-tainer manufactured by Fujimori Industry Co., Ltd. Since this inner bag has a spout (filling port), it is possible to store and discharge the medium, take out the culture, and enclose the liquid in a liquid-tight manner, without using a heat welder or the like. It is preferable because it can be sealed with a lid member.

As the base material of the bottomed packaging bag that becomes the main body of the culture bag, it is flexible, water-impermeable, and has high strength, so that it is a synthetic resin film or sheet (in this specification, these are collectively referred to as “film”) .) Is preferred. And from a viewpoint of manufacturing a culture bag, what has heat-welding property is preferable, and what can see through the inside of a container from the workability | operativity of culture | cultivation is preferable.
Examples of the synthetic resin constituting such a film include polyolefins such as polyethylene (PE) and polypropylene (PP), and soft vinyl chloride. When particularly high strength is required, a biaxially stretched nylon film or a biaxially stretched polyethylene terephthalate (PET) film may be laminated as a reinforcing layer. When gas barrier properties such as oxygen are required, a film in which a vapor deposition layer of silica or alumina, an ethylene-vinyl alcohol copolymer layer, or the like is laminated may be used. A plurality of these films may be laminated or superposed.

The thickness of the base material which comprises a packaging bag is not specifically limited, According to the material of a base material and the capacity | capacitance of a culture bag, it can select suitably. The culture bag of the present invention preferably has a certain degree of elasticity from the viewpoints of workability when being supported by a support device and shape retention during culture, and is usually preferably about 50 μm to 1000 μm.
Although the capacity of the culture bag is not particularly limited, it can be set to, for example, a capacity of 5 to 1000 L.

  The culture bag of the present invention includes an air supply pipe to which a weight is added. The air supply pipe communicates with the inside and outside of the culture bag and supplies gas into the culture bag from the outside. It is possible to perform the desired culture by supplying air from the supply pipe to aerobic conditions by supplying air or by supplying an inert gas such as nitrogen gas or argon. A weight is added to the air supply pipe so as to be able to settle in the lower part of the culture bag against the buoyancy caused by the gas in the culture medium. The location where the inside and outside communicate with each other through the air supply pipe is not particularly limited, but the upper part of the front surface (the front or back main surface film) of the packaging bag is preferable.

  The air supply pipe can be installed even if a hole is provided in the packaging bag, the air supply pipe is inserted directly into the hole to allow communication between the inside and outside of the packaging bag, and the insertion point is sealed liquid-tight with adhesive or heat welding. As in the case of the Z-tainer, a hole is formed in the upper part of the packaging bag, and a synthetic resin filling port (outlet) protruding outside is thermally welded from the inside and sealed liquid-tightly with a synthetic resin lid. Preferably, the lid is provided with a communication member that communicates the inside and outside of the lid, and an air supply pipe is connected to the lid so as to communicate with the inside and outside of the packaging bag.

In order to connect the air supply pipe to the inside and outside by providing a communication member on the lid, for example, a hole is made in the lid in advance, and one or a plurality of pipes or nozzles having a flange as the communication member are arranged inside and outside the lid. The flange and the lid may be liquid-tightly sealed with an adhesive or heat welding. Alternatively, a hole may be formed in the lid, and a pipe serving as a communication member may be passed through the hole, and the penetration portion may be sealed in a liquid-tight manner. Moreover,
The communication member may be formed inside and outside the lid when molding the lid. It is preferable to provide a plurality of communication members for such a lid, and to these communication members, not only an air supply pipe but also an exhaust pipe and a pipe for monitoring and sampling the pH and temperature of the culture medium are connected. Can do.

  The air supply and exhaust pipes that extend outside the culture bag are fitted with a filter to prevent contamination by germs at the middle or end, or fixed with an adhesive or heat welding, and sealed. This is preferable because the gas can flow in and out while maintaining the sterile condition in the culture bag, and the remaining air in the culture bag can be expelled when the culture bag is sealed. The filter may be installed at an intermediate portion or an end portion of the communication member protruding outside the lid.

There is no particular limitation on the material of the air supply pipe, but it is preferable to have flexibility since it does not damage the packaging bag. A metal flexible tube may be used as long as it has flexibility, but a synthetic resin tube is preferable because it is lightweight. In particular, in the present invention, since a weight is added, a PE or PP pipe having a low density can be used.
The length of the air supply pipe is set so that a part of the air supply pipe is in contact with the bottom surface of the culture bag when the culture bag is made independent. Preferably, it is set to lie on the bottom with a predetermined length.

  It is preferable to provide a plurality of air supply holes in a part of the culture bag of the air supply tube so that bubbles can be generated (bubbling). As the air supply hole of the air supply pipe, the opening at the tip in the culture bag may be used as the air supply hole as it is, but it is preferable to provide a plurality of holes along the air supply pipe so as to efficiently bubble. In this case, the air supply hole is preferably provided at a position deeper than at least the position where the culture solution is assumed to exist, and the tip of the air supply tube in the culture bag is heat-welded or by appropriate means such as a pinch cock or a stopper. It is more preferable to seal.

  The weight is added near the tip of the air supply pipe so that the portion having the air supply hole of the air supply pipe settles in the culture medium. Preferably, a weight is added so that a part of the air supply pipe contacts the bottom surface of the culture bag when the culture bag is made to stand. The weight may be connected to or fixed to the tip of the air supply tube in the culture bag. However, when there are a plurality of air supply holes, along the air supply tube, the portion having the plurality of air supply holes lies on the bottom surface. It is preferable to install a bar or a plurality of weights. In this case, for example, a rod-like weight is fixed with a fastener along the air supply pipe so as not to block the air supply hole, or a rod-like weight having an inner diameter smaller than the inner diameter of the air supply pipe is inserted into the air supply pipe. It is preferable to seal the end of the trachea. The material of the weight is not particularly limited as long as it can be precipitated in the culture medium, but preferably has high corrosion resistance, and a metal such as stainless steel is preferable.

  The culture bag of the present invention further includes a liquid supply pipe for injecting microorganisms to be cultured, a sample collection, a circulation of the culture liquid, a drainage pipe for discharging excess or unnecessary liquid, and a gas discharge. An exhaust pipe or the like may be installed. These may be installed directly on the culture bag, but it is preferable to provide a communication member on the lid as in the case of the air supply pipe and install it through this.

  The culture bag of the present invention may be provided with a hanging part on the upper part of the packaging bag so that the culture bag can be supported on the support device by this hanging part. The hanging part may be a string or hook fixed to the upper edge of the culture bag with an adhesive or the like. However, if a hole for hooking is provided in the upper part of the packaging bag in advance, Can be installed easily. Specifically, there may be mentioned a method in which both upper corner portions of the packaging bag are thermally welded obliquely, separated from the storage portion, and holes are formed in the corner portions.

Depending on the nature of the microorganism to be cultured, the inside of the culture bag may not necessarily be in a sterile state, but it is usually preferable to keep it in a sterile state. For example, the culture bag of the present invention can be manufactured in a clean environment or sterilized after the culture bag is manufactured to sterilize the inside. The method for sterilizing the culture bag is not particularly limited. For example, radiation sterilization such as γ-ray sterilization, ethylene oxide gas (EOG) sterilization, autoclave (AC
) Sterilization and the like. Of these, AC sterilization is preferred because the device is simple and easy to operate. When performing EOG sterilization, it is preferable to use at least a part of the container body as a filter. In this case, it is preferable that the filter is a polyolefin-based non-woven fabric such as PE or PP because heat welding is possible. In addition, you may sterilize, after folding the culture bag of this invention and accommodating in an exterior bag or an exterior box. A plurality of culture bags may be folded and housed together in a single outer bag or outer box and sterilized. For example, in the case of performing aseptic connection with a communicating member on a clean bench or the like, It is preferable to sterilize them one by one in an outer bag or outer box.

  The culture bag of the present invention is used with the bottom surface supported by a support device. The support device preferably has a mounting surface that can support the bottom surface of the culture bag and can support the upper portion of the culture bag, and is attached to the support device to suspend the culture bag. It is more preferable to have The frame preferably has a clip or hook like a clothespin, and if the culture bag has a hanging part, the hook can be easily and stably hooked on the hook of the support device. More preferably.

  The support device preferably includes a swing device that can swing in the horizontal direction. By mounting the culture bag of the present invention on a support device having a mounting plate that can swing in the horizontal direction, it can be used as a shaking culture device. The support device having a shaking function can be obtained by installing a mounting plate on a commercially available shaker. In order to prevent the culture bag from slipping down on the mounting plate, it is preferable that a wall is erected around the mounting plate to form a plate or a box. Moreover, when the dish or box is remarkably larger than the bottom surface of the culture bag, it is preferable to use an adhesive or a gel-like stabilizer at the bottom so as not to shift during shaking. Examples of commercially available shakers include a reciprocating shaker manufactured by TAITEC.

  In the culture apparatus of the present invention, a temperature sensor may be installed in order to monitor and control the temperature of the culture solution or culture in the culture bag. As the temperature sensor, a contact type such as a thermocouple or a thermistor or a non-contact type such as a radiation thermometer can be used. The temperature sensor may be directly attached to the culture bag or may be attached to the support device. Further, a temperature sensor may be connected to the drainage pipe so that, for example, the culture medium being cultured can be sucked through the drainage pipe and the temperature of the sucked culture medium can be detected.

  In addition, for example, in the culture of mammalian cells such as CHO cells and hybridomas, the pH, oxygen concentration, etc. of the culture solution may be monitored by a sensor instead of the temperature or in combination with the temperature, if necessary. In this case, it is desirable to perform sensing by connecting a tube or the like aseptically to the communication member of the lid and performing sampling or circulating a part of the culture solution outside the culture bag such as a clean booth.

  The culture apparatus of the present invention is preferably provided with a heating apparatus in order to warm the inside of the culture bag and promote culture. The warming device may be installed on the side surface of the culture bag or on the bottom surface. Or you may install in both. Although the heating device may be fixed to the culture bag, for example, the heating device may be provided only on the surface of the mounting tray of the support device. As the heating device, a heating element such as a sheet heating element in which a circuit of nichrome wire, sheathed wire, carbon or aluminum foil is sandwiched between synthetic resin layers can be used. This heating device is preferably controllable based on information from a temperature sensor attached to the culture device.

The culture apparatus of the present invention can be used not only for culturing microorganisms but also for producing substances using microorganisms.
Moreover, after culturing microorganisms or the like with the culture apparatus of the present invention, the microorganisms or the like can be immobilized on a carrier in a culture bag to form a bioreactor. For example, after culturing microorganisms in a culture bag, an appropriate amount of a carrier for immobilizing microorganisms may be added to the culture bag, and the microorganisms may be immobilized while stirring with a stirrer or a shaker. At the time of immobilization, aerobic conditions are introduced by introducing air from the air supply pipe while stirring in the culture bag of the present invention, or anaerobic conditions are introduced by introducing an inert gas such as nitrogen gas or argon. It is possible to carry out the desired reaction. In this case, the carrier used is not particularly limited. For example, Kuraray manufactured by Kuraray Co., Ltd. or APG manufactured by Nisshinbo Co., Ltd. can be used.

  The culture bag of the present invention contains a medium, preferably a liquid medium, and is sterilized by, for example, AC or retort, and is easy to carry if it is left as it is or if one bag or several bags are put in an outer box. Can not be propagated and can be transported far away. In addition, after accommodating a culture bag in an exterior bag, it may be transported in an exterior box. The cultivator who has received the transported medium in this way can perform the culture simply by inoculating the microorganism in the culture bag, and does not need to prepare the medium or perform sterilization by himself. is there. In addition, since the culture bag of the present invention can be transported to a predetermined place without removing the cultured body after completion of the culture, it can be easily transferred to the next step such as collection of the target substance. it can.

Hereinafter, the first embodiment of the present invention will be described in detail with reference to the drawings.
FIG. 1 is a plan view of the culture bag 10 of the present invention in a folded state. FIG. 2 is a perspective view of the culture bag 10 of the present invention in an inflated state.
The culture bag 10 of the present invention shown in FIGS. 1 and 2 is a gusset-shaped BIB inner bag disclosed in Japanese Patent Application Laid-Open No. 2003-191967, and the specification of a Z-tainer manufactured by Fujimori Kogyo Co., Ltd. having a nominal capacity of 20 L is partially changed. The thing is used as a bottomed packaging bag.

  This packaging bag is provided with a circular handle hole having a diameter of 40 mm, with the corners on both sides of the upper part separated from the storage part by an oblique welded part having a width of 10 mm. This handle hole can be used when the culture bag 10 is supported. The front film is provided with a hole having a diameter of 45 mm, and a PP spout (filling port) having an inner diameter of 32 mm having a flange with a width of 15 mm is heat-welded. The cylindrical portion of the spout is provided with an external screw, and a PP cap having an internal screw is attached to form a set.

  The substrate was a PP film single layer having a thickness of 130 μm, and the size was 580 mm in length, 320 mm in width, 140 mm in gusset folding, and the periphery was thermally welded with a width of 10 mm. In general, BIB inner bags are often used with PE film in consideration of pinholes and drop strength during transportation, and two films are used in layers, but in this embodiment, special consideration is given during transportation. Instead, considering the AC sterilization and retort sterilization described later, the base material was a PP film, and the film was also a single sheet. If both are taken into account, for example, two 100 μm PP films can be used, but the cost increases.

  The handle hole at the top of the Z-tainer was used as the hanging portion 17 and the attached cap was used as the lid 14. When the cap was used as the lid 14, a hole having a diameter of 30 mm was provided on the top surface of the cap. Insert a disc-shaped inner lid that is integrally molded so that the three PP tubes sealed at the outer end when used as the communication member protrude from the front and back sides. It was set as the lid | cover 14 which adhere | attached liquid-tightly with the edge of an opening and an adhesive agent, and has a communicating member.

A silicone rubber tube was externally fitted to one of the communicating members of the lid 14 facing the inside of the packaging bag to form an air supply pipe 11. The communication members 12 and 13 are externally fitted with silicone rubber tubes, and supply pipes for injecting microorganisms, culture solutions, etc., and circulation of exhaust, sample collection, culture solution, etc., or excessive or unnecessary. Used to connect a drainage pipe that discharges the liquid.
The supply tube 11 is sealed at the tip in the culture bag, and has a plurality of holes opened along the portion in contact with the bottom surface of the culture bag. A stainless steel rod-like weight 15 is fixed to the air supply pipe 11 with two fasteners 16 along the portion of the air supply pipe 11 having the air supply holes.

The spout of the gusset packaging bag is liquid-tightly sealed or sealed with a lid 14 having a communication member connected to the air supply pipe 11, a supply pipe connection communication member 12, and a drainage pipe connection communication member 13. The culture bag 10 of the present invention is obtained as a set without stopping.
In the present embodiment, the spout of the obtained culture bag 10 was sealed in a liquid-tight manner with the lid 14 and further sterilized with γ rays, and the entire interior was made sterile.

Next, a second embodiment of the present invention will be described.
FIG. 3 is a perspective view of the culture apparatus 100 of the present invention. The culture apparatus 100 shown in FIG. 3 has the following configuration. First, a shallow box-shaped stainless steel mounting plate 32 in which the lower part of the culture bag 10 was placed was installed on the top surface of the support device 20 having a swinging device that swings in the horizontal direction by combining a motor and a crank. And the planar heater which is not illustrated was spread | laid on the bottom face of the mounting board 32, and the culture bag 10 obtained by the 1st embodiment was mounted. In addition, the heater which coat | covered the circuit of the nichrome wire with the silicone rubber was used for the planar heater. Other planar heaters that can be used include an aluminum foil circuit sandwiched between synthetic resin sheets and carbon enclosed in a packaging bag.

  The frame body 30 heading upward is attached to the left and right outer wall surfaces of the mounting plate 32. The culture bag 10 was supported by providing hooks 31 at two horizontal bars on the upper portion of the frame 30 and hanging the hooks 31 on the hanging portions 17 (handle holes) of the culture bag 10. The height of the frame 30 is set slightly lower than the height of the culture bag 10 so that the front and back films can be extended without the culture bag 10 being suspended, and the bottom surface of the culture bag 10 is in contact with the heater. did. By supporting in this way, even if the inside of the culture bag 10 is not at a positive pressure, the culture bag 10 will not be bent due to the weight of the spout, the lid 14 or the air supply pipe 11, so that a sufficient internal space can be obtained. Can be secured.

Next, the usage method of the culture apparatus 100 of this invention is demonstrated using FIG.
Silicone rubber having a sterilized culture bag 10 with an end facing the outside of the communication member connected to the air supply pipe 11 open in a clean bench, the inside and outside being kept in a sterile state, and a filter (not shown) at the end A manufactured air supply tube was fitted on the outside of the culture bag 10 to extend the air supply tube. Similarly, a silicone rubber liquid supply tube (not shown) is externally fitted to the outer end of the communication member 12 to form a liquid supply pipe, and a culture liquid container containing a sterilized culture liquid (not shown) is stored at the end. Connected aseptically. Similarly, a silicone rubber exhaust tube having a filter (not shown) is externally fitted to the outer end of the communication member 13 inside and outside, and an exhaust pipe is formed by fitting a filter (not shown) on the end.

The culture bag is filled with the culture solution in the culture solution container via the supply tube, and the culture solution container is removed from the clean bench and filled with the culture solution via the supply tube in order to prevent contamination of bacteria. 10 was injected with a culture such as a microorganism.
The culture bag 10 containing the culture solution and the culture was placed on a shallow stainless steel container (mounting plate 32) of a reciprocating shaker (supporting device 20) manufactured by TAITEC. An air compressor is connected to the filter of the extended supply pipe, and the air filtered through the supply pipe 11 is pumped into the culture bag 10 and supplied to the supply pipe 11 that is submerged in the culture medium. Bubbles were generated from the pores. The oscillating device of the support device 20 was started, the mounting plate 32 was oscillated in the horizontal direction, and shaking culture was performed while supplying air.

The temperature inside the culture bag 10 is measured by a temperature sensor (not shown) installed on the inner wall surface of the mounting plate 32, and the inside of the culture bag 10 is provided below the bottom surface of the culture bag 10 based on the information of the temperature sensor. The temperature was controlled at a predetermined temperature with a heater coated with silicone rubber (not shown). If necessary, aseptically remove the exhaust pipe from the drainage tube connection communicating member 13, fit the suction tube and suck it with a pump, collect the sample and check the culture concentration, The culture was terminated when the culture reached a predetermined concentration.

  When the culture is completed, the culture bag 10 may be opened and the culture body may be recovered. In this embodiment, the culture body is not recovered, but connected to the communication member of the lid 14 of the culture bag 10 in the clean booth. All the outer tubes were removed, and the outer ends of all the communicating members were sealed with heat welding, pinch cocks, etc., and the culture bag 10 was sealed. Thus, the culture medium accommodation culture bag of the present invention can be obtained. In this case, the end portion and the intermediate portion may be similarly sealed without removing the tube.

By sealing in this way, the culture bag 10 containing the culture is placed in a cardboard outer box having a vertical, horizontal, and height of 290 × 290 × 260 mm (inner dimensions) that is commercially available as an outer package of the Z-tainer. Or stored in a normal cardboard box, and the culture can be transported to a predetermined place without being refilled into another transport container.
And when using the Z-tainer outer box, the perforation is formed in the upper flap so that the spout opening door and the fixing hole can be opened. The hole and door can be opened, the lid 14 can be gripped and pulled out together with the spout, and the door can be closed to fix the spout. Thereby, content items, such as a culture body in the culture bag 10, can be poured out without opening a cardboard box.

Next, a third embodiment of the present invention will be described.
In this embodiment, after the culture bag 10 obtained in the first embodiment is filled with the medium, the culture bag 10 is sterilized together with the medium.
That is, the lid 14 having the communication members 12 and 13 whose outer end portions are sealed, and the air supply pipe 11 connected to the inner end portion of the communication member whose outer end portions are sealed, and the packaging serving as the main body of the culture bag 10 The culture bag 10 is filled with the culture medium from the filling port of the culture bag 10 in which the bag is set, and the culture bag 10 is liquid-tightly sealed with the lid 14 and then sterilized. In this case, the inside of the culture bag 10 filled with the medium may not be sterilized in advance. Thus, the culture medium accommodation culture bag of the present invention can be obtained.

Since the inside of the culture bag containing culture medium is maintained in a sterilized state by sterilization, it can be cultured simply by inoculating the organism into the culture bag 10 containing the culture medium.
In addition, since the sterilization method of the culture bag 10 in which the medium is accommodated needs to be sterilized to the inside of the medium, heat sterilization such as γ-ray sterilization, retort sterilization, AC sterilization, and the like can be given. Among these, retort sterilization is preferable because heat sterilization is simple, rapid cooling is possible, and since there are many achievements in such sterilization, the reliability is high and the apparatus is complete. Retort conditions vary depending on the properties of the medium, but if the properties of the medium and the type of bacteria to be sterilized are known, they can be easily set by testing with an F value monitor.

The culture examples of the present invention will be given and further described below.
Except that three PP nozzles (communication members) are fixed to the top surface of the spout lid 14 with an adhesive so that outside air does not enter from other than the nozzles, the same as in the first embodiment. Thus, a culture bag 10 whose interior is not sterilized as shown in FIG. 1 was produced.

Escherichia coli HB101 strain was used as an indicator microorganism in this example. 1 ml of E. coli strain HB101 stored as a glycerol stock was inoculated into 100 mL of LB medium (tryptone 1%, yeast extract 0.5%, NaCl 1%), followed by overnight shaking culture (preculture). 10 L of LB medium was injected into the culture bag 10 for AC sterilization. If the device is opened while the inside of the device is hot, the culture bag may break, so allow enough time for natural cooling and prevent contamination with germs. The culture bag 10 was poured and seeded.

  Thereafter, the culture apparatus 100 of the present invention shown in FIG. 3 was configured in the same manner as in the second embodiment. The culture bag 10 into which the culture body was injected was accommodated in a shallow stainless steel container (mounting plate 32), and the culture bag 10 was supported by the mounting plate 32 and the hook of the frame 30. The air supply tube 11 was supplied with an air compressor, and cultured with shaking while performing reciprocal shaking at 70 rpm with a reciprocating shaker. As a comparative experiment, 100 ml of LB medium was dispensed into a 500 ml flask and a 500 ml baffle flask, respectively, 1 ml of the above pre-cultured culture was seeded, and shaking culture was performed at 37 ° C. and 200 rpm.

After the start of the culture, sampling was performed at an interval of 1 hour, and the growth was confirmed by measuring the turbidity (OD600) (the higher the OD600 value, the more the microorganism was growing).
The results are shown in FIG.
As shown in FIG. 4, it was shown that the culture bag of the present invention showed a slight growth even when compared with existing flasks and baffle flasks with large oxygen uptake. Furthermore, it is expected that higher yields can be obtained by changing the medium composition or directly bubbling oxygen.

  As mentioned above, although this invention was demonstrated based on some embodiment, this invention is not limited to this, A various change is possible within the scope of the present invention. For example, four or more communication members may be provided. One may be dedicated to sampling and monitoring. In this case, like the installation of the air supply pipe, it is preferable to connect the extending tube to the inner end of the communication member so as to have an appropriate depth. With such a configuration, a part of the culture solution can be circulated outside the culture bag such as a clean bench to monitor the temperature, pH, turbidity, oxygen concentration, etc. of the culture solution.

  The present invention can be used for a culture bag or a culture apparatus that can easily and inexpensively perform mass culture of microorganisms.

The top view of the state which folded the culture bag of this invention. The perspective view of the state which the culture bag of the present invention expanded. The perspective view of the culture apparatus of this invention. The figure which shows the growth curve of colon_bacillus | E._coli (HB101) when it culture | cultivates with the culture bag of this invention, a 500 ml flask, and a 500 ml baffle flask.

Explanation of symbols

10. Culture bags, 11. Air supply pipe, 12. 12. Communication member (for liquid supply pipe), Communicating member (for drainage pipe), 14. Lid, 15. Weight, 16. Fasteners, 17. Hanging part, 20. Support device, 30. Frame, 31. Hook, 32. Mounting plate, 100. Incubator

Claims (14)

A culture bag comprising a bottomed packaging bag capable of containing liquid in a liquid-tight manner and capable of being self-supporting when the liquid is contained, and comprising a supply pipe with a weight added in the culture bag. The culture bag according to claim 1, wherein a weight is added so that a part of the air supply pipe contacts the bottom surface of the culture bag when the culture bag is made to stand. The culture bag according to claim 2, wherein the air supply pipe has a plurality of air supply holes, and a bar-like weight is added along the air supply pipe so that a portion having the plurality of air supply holes lies on the bottom surface. The culture bag according to any one of claims 1 to 3, wherein the packaging bag is a gusset bag. The culture bag according to any one of claims 1 to 4, wherein the packaging bag includes a hanging portion at an upper portion. A culture device comprising the culture bag according to any one of claims 1 to 5 supported by a support device that supports at least a bottom surface of the culture bag. The culture device according to claim 6, wherein the support device further supports an upper portion of the culture bag. The culture apparatus according to claim 6 or 7, wherein the support device includes a swinging device that can swing in a horizontal direction. The culture device according to any one of claims 6 to 8, wherein the culture device includes a temperature sensor. The culture device according to any one of claims 6 to 9, wherein the culture device includes a heating device. A method for culturing cells or microorganisms, wherein gas is supplied from the air supply pipe using the culture bag according to any one of claims 1 to 5 to culture cells or microorganisms. The method for culturing cells or microorganisms according to claim 11, wherein the culture bags are shaken to culture the cells or microorganisms. A culture medium-containing culture bag in which the culture medium is sealed in the culture bag according to any one of claims 1 to 5 and maintained in a sterile state. A method for transporting a culture, wherein the culture cultured using the culture bag according to claim 1 is transported without being taken out of the culture bag.

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