JP4986659B2 - Culture bag and incubator - Google Patents

Description

  The present invention relates to a culture bag and an incubator capable of culturing cells and microorganisms in large quantities.

Mass 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 large incubators made of glass or stainless steel are expensive, and a large sterilizer is required to sterilize the incubator, or a sterilizer is attached to the incubator itself. Equipment was needed. 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 during assembly of the incubator or storage of the culture medium or culture medium. As a result, there was a limit to maintaining the sterility inside the incubator.
On the other hand, a culture bag having a sealed structure for culturing anaerobic microorganisms has also been proposed (Patent Document 2). This culture bag is suitable for maintaining the aseptic condition inside the incubator, but it has a flat bag structure in which two rectangular sheets are welded on four sides, and stirring the inside of the culture bag is considered. Not.
Japanese Patent Laid-Open No. 06-153902 JP 2006-25617 A

  The present invention has been made in order to solve the above-described problems. The inside of the present invention is configured so that it can be stirred, and is used for mass culture of cells and microorganisms (hereinafter collectively referred to as “microorganisms”) and various types of microorganisms. It is an object of the present invention to provide a culture bag and an incubator that can perform the above operation inexpensively and simply.

  As a result of intensive studies to solve the above-described problems, the present inventors have solved the problem by housing a stirring device having a rotating shaft having a stirring blade in a bottomed and covered cylindrical container made of a foldable base material. As a result, the present invention has been completed.

That is, the present invention is as follows.
(1) A culture bag in which a stirrer having a rotating shaft having a stirring blade is hermetically housed in a foldable bottomed and covered cylindrical container, and the upper edge of the container is a suspension made of an extension of the trunk wall A culture bag in which a throat is provided over the entire circumference and a spout that can be hermetically sealed with a cap is thermally welded to the lid of the container .
(2) The culture bag according to (1), wherein the spout has a plurality of heat-welded lids.
( 3 ) The culture bag according to (1) or (2) , wherein the agitation apparatus is stored in an airtight manner by projecting the rotating shaft from the spout, which is thermally welded to the lid of the container, so as to be rotatable outside the container.
( 4 ) The container has a tubular communication member that communicates the inside and outside of the container with one end projecting to the outside of the container sealed in the cap to hermetically seal the spout. The culture bag according to any one of (1) to (3), which is airtightly arranged.
( 5 ) The culture bag according to any one of (1) to ( 4 ), wherein the container includes a temperature sensor on the outside of the trunk wall.
( 6 ) The culture bag according to any one of (1) to ( 5 ), wherein the inside of the culture bag is maintained in a sterile state.
( 7 ) An incubator in which the culture bag of any one of (1) to ( 6 ) is housed in a rigid outer shell. ( 8 ) The incubator according to ( 7 ), wherein the outer shell is a cylinder.
( 9 ) The incubator according to ( 7 ) or ( 8 ), wherein the incubator includes a drive device that rotates the rotating shaft.
( 10 ) The incubator according to any one of ( 7 ) to ( 9 ), wherein the incubator includes a heating device.
( 11 ) The incubator according to any one of ( 7 ) to ( 10 ), wherein the incubator includes a cooling device.
( 12 ) The incubator according to any one of ( 7 ) to ( 11 ), wherein the incubator includes a pH adjusting device. ( 13 ) The incubator according to any one of ( 7 ) to ( 12 ), wherein the incubator includes an air supply device.
( 14 ) The incubator according to any one of ( 7 ) to ( 13 ), wherein the incubator includes a sensor for measuring one or more of pH, temperature, dissolved oxygen, and turbidity in the culture bag.
(15) incubator of the provided pH of the incubator is in the culture bag on the basis of information of the sensor, temperature, a control device for controlling one or two or more of the dissolved oxygen and turbidity (14).

The culture bag of the present invention is a closed-bottomed cylindrical container that can fold the bag body, so that the volume can be reduced during storage and transportation, and the inside can be stirred during use. Various operations can be performed inexpensively and easily. In addition, since the stirrer is housed in a foldable bottomed and covered cylindrical container in an airtight manner, for example, the culture bag of the present invention can be produced in a clean environment or sterilized in advance with γ-ray or the like. Thus, by maintaining the sterilized state, it is possible to easily carry out the culture without performing a time-consuming sterilization operation. Moreover, the culture bag after use becomes disposable, and washing | cleaning of the incubator after use is unnecessary. And since a container is cylindrical shape, a stirring blade does not contact a container trunk | drum wall at the time of stirring operation. In addition, it is possible to avoid that the bottom of the culture bag cannot be sprinkled and the culture body is clogged with the sputum and the culture efficiency is lowered.
In addition, when the container is provided with a communication member, the culture can be performed by aseptically connecting a container in which a culture solution or a culture body is stored while a tube is connected or can be supplied and exhausted as it is. When the culture solution or culture is stored and taken out in the vessel, no germs are mixed.
Further, when the container is provided with a temperature sensor on the outside of the body wall, temperature management can be performed without contacting the stirring device.
Moreover, when the said container is provided with a suspension part on the upper edge, it can be reliably and easily fixed to the support member. And when the said suspension part is the extension part of the trunk | drum wall of the said container, a suspension part can be easily installed at the time of manufacture of a container. Further, when the outer shell is a cylinder, the container can be easily fixed to the outer shell by folding the hanging portion.
Since the culture bag of the present invention is housed in the outer shell having rigidity, the incubator of the present invention can be made to stand by itself stably. And if the outer shell is a cylinder, it is highly versatile and can be easily used steel drums, synthetic resin drums, paper composites, etc., and the incubator can be made compact. .
If the incubator includes a drive device that rotates the rotating shaft, more stable agitation culture is possible.
Further, when the incubator is equipped with a heating device, the culture can be promoted by heating.
In addition, when the incubator is provided with a cooling device, substance production and biological reaction in the culture bag can be terminated at a specific timing.
Further, when the incubator is equipped with a pH adjusting device, stable culture can be performed while keeping the pH constant, and the target substance can be precipitated by changing the pH of the culture solution.
In addition, when the incubator includes an air supply device, efficient aeration culture is possible.
In addition, when the incubator includes a sensor for measuring one or more of pH, temperature, dissolved oxygen and turbidity in the culture bag, stable culture can be performed while monitoring pH, temperature, dissolved oxygen and turbidity. It becomes possible.
Further, when the incubator includes a control device for controlling one or more of pH, temperature, dissolved oxygen and turbidity in the culture bag based on the information of the sensor, the pH, temperature, dissolved oxygen and turbidity are controlled. Control becomes easy.

The culture bag of the present invention comprises a foldable base material. The base material that can be folded is preferably a synthetic resin film or sheet (in this specification, “film” and “sheet” are collectively referred to as “film”) because it is flexible, impermeable, and has high strength. . 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 film may be laminated as a reinforcing layer. When the container is required to block oxygen, a gas barrier substrate such as a metal foil such as aluminum, an inorganic vapor deposition film, or an ethylene-vinyl alcohol copolymer layer may be used. A plurality of these films and base materials may be laminated or superposed.
The thickness of the substrate is not particularly limited and can be appropriately selected according to the material of the substrate and the capacity of the container. The culture bag of the present invention is foldable, and preferably has a certain degree of waist from the viewpoint of workability when assembling into an incubator and shape retention during culture, and is usually about 50 μm to 1000 μm. .

  A culture bag can be produced by molding the base material as described above into a cylindrical container with a bottom and a lid. Even when the culture bag is made into a cylinder with a bottomed lid, it can be avoided that the bottom of the culture bag is not crushed and the culture body is clogged with the culm and the culture efficiency is lowered. Further, it is possible to avoid a stirring blade or the like from coming into contact with the body wall during stirring. Although the capacity | capacitance in particular of a container is not restrict | limited, For example, it can be set as the capacity | capacitance of 5-1000L. When used as a large-scale incubator of about 200 L, “Fuji Liner 200” manufactured by Fujimori Kogyo Co., Ltd. has a spout (filling port) that can be sealed with a cap on a commercially available cylindrical drum can inner bag, for example, the top surface (lid). The specification of “can be partially changed and used.

Inside the cylindrical container, a stirrer including a rotating shaft having a stirring blade is housed in an airtight manner. When the rotating shaft is directly driven by an external motor or the like during storage, the rotating shaft protrudes from the container lid to the outside of the container so as to be rotatable using an airtight bearing, etc. Store in. Moreover, when driving indirectly using a magnet, the rotating shaft is stored in an airtight manner without protruding from the container lid to the outside of the container.
Examples of the stirrer include a stirrer in which a stirrer blade is fixed to the tip and / or middle of a rotating shaft made of metal or synthetic resin. It is preferable that two stirring blades are fixed with the rotating shaft between them so that the culture bag can be easily folded. The stirring blades may be provided in a plurality of stages. In this case, you may shift the angle of the attachment position of the blade | wing of each step | level. When fixing three or more sheets, it is preferable that the stirring blade be foldable. When the width of the stirring blade is wide, it is preferable that the horizontal twist angle can be adjusted. When the folded stirring blade is expanded or the twist angle is adjusted, the base material has flexibility, so that it can be operated from the outside of the container through the base material.

The cylindrical container is easily connected to a tube made of synthetic resin with an arbitrary length by providing an airtight tubular communication member that communicates the inside and outside of the container with one end projecting outside the container sealed. This is preferable. Specifically, the communication member is installed, for example, by providing a hole in the lid of the cylindrical container, and directly inserting the communication member into the hole to allow communication between the inside and the outside of the cylindrical container. Seal liquid-tightly. Alternatively, a pipe having a flange is previously arranged inside and outside the hole provided in the upper wall of the container and the lid, and the flange and the base material of the container are hermetically fixed with an adhesive or heat welding, and then the container The body wall and the lid are hermetically fixed with an adhesive or heat welding. Furthermore, like the above Fuji liner, a synthetic resin spout member having a hole in the top surface of the cylindrical container and having a flange from the inside is inserted, and the spout port protruding outward is thermally welded with a synthetic resin cap. It is preferable that the cap is provided so as to be capable of being sealed in a liquid-tight manner, and a communication member that communicates the inside and outside of the cap is provided.
In order to make the cap communicate with the inside and outside by providing a communication member, for example, a hole is made in the cap in advance, and one or more tubes or nozzles having a flange as the communication member are arranged inside and outside the cap. The flange and the cap may be liquid-tightly sealed with an adhesive or heat welding. Alternatively, a hole may be formed in the cap, 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. Further, the communication member may be formed inside and outside the cap when the cap is molded. The tube and the nozzle may be extended inside the container. Airtightness can be ensured by preliminarily sealing one end of a tube and nozzle that are outside the container, or by sealing by heat welding at the time of fixation. It is preferable to provide a plurality of communication members because a liquid supply pipe, a liquid discharge pipe, an air supply pipe, an exhaust pipe, and the like can be provided. When connecting a tube to a communication member to make an air supply pipe or an exhaust pipe, fit a filter to prevent contamination of bacteria on the end of the communication member or tube, or fix it with an adhesive or heat welding. Sealing is preferable because gas can flow in and out while maintaining the aseptic condition in the container, and the remaining air in the container can be expelled when folded.

A temperature sensor may be installed outside the body wall of the container in order to monitor and control the temperature of the culture solution or culture in the cylindrical container. The temperature sensor may be installed inside the container via the communication member, but it is preferable to install the temperature sensor outside the cylindrical container because contamination can be prevented. Moreover, it does not contact a stirring blade at the time of stirring. 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. In the case of a radiation thermometer, the temperature sensor may be provided on the outer shell.
In addition, for example, in the culture of mammalian cells such as CHO cells and hybridomas, the pH, turbidity, oxygen concentration, etc. may be monitored by a sensor instead of temperature or in combination with temperature as necessary. In this case, it is desirable to aseptically connect a tube or the like to the communicating member and perform sampling, or to directly sense the culture solution by circulating a part of the culture solution outside the incubator such as a clean booth. And it is desirable to process the result of sensing with a computer etc., and to control 1 type, or 2 or more types of pH, temperature, dissolved oxygen, and turbidity in a culture bag.

  When the container is provided with a hanging part on the upper edge, the container can be easily supported by the outer shell as a supporting member by this hanging part. The hanging part may be attached to the outer wall of the container with an adhesive or the like, but if the hanging part is an extension of the body wall of the container, the hanging part can be easily installed when the container is manufactured. it can. Moreover, you may provide an eyelet etc. here. When the suspension part consisting of the extension part of the body wall is provided over the entire circumference, the container can be easily fixed to the outer shell by folding the suspension part when the outer shell is a cylinder. it can.

  The inside of the culture bag does not necessarily need to be sterilized depending on the nature of the culture, but is usually preferably maintained in a sterilized state. The aseptic condition can be aseptically prepared by, for example, producing the culture bag of the present invention in a clean environment or sterilizing in advance. The method for sterilizing the culture bag is not particularly limited, and examples thereof include radiation sterilization such as γ-ray sterilization, ethylene oxide gas (EOG) sterilization, and autoclave (AC) sterilization. When performing EOG sterilization, it is preferable to use at least a part of the container 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 fusion is possible.

Cylindrical containers show a certain degree of independence when a large amount of culture medium or culture medium is stored and the head space is small. It is preferable to ensure the shape retention of the container using a shell. The shape of the outer shell is not particularly limited, and a frame, a box, a cylindrical can, or the like in which a column or a tube is assembled can be used. These may be bottomless, but are preferably bottomed because the culture bag can be supported from below. By housing in the outer shell of the cylinder, the culture bag of the present invention can be stably supported, and a cylinder is preferable because the incubator can be made compact. If the inner diameter of the outer shell of the cylinder is about the same as or slightly smaller than that of the culture bag, the culture bag will be in close contact with the outer shell when filled with the culture solution, etc. It is preferable because it can be stably stirred.
The material of the outer shell is not particularly limited as long as it has sufficient rigidity to hold a culture body to be cultured or a culture solution or culture bag filled with the culture solution, and examples thereof include metals, plastics, and paperboard. Durability, particularly when the incubator is heated, is preferably a metal. In consideration of the occurrence of rust when repeatedly used, stainless steel is more preferable. When the material of the outer shell is opaque, a viewing window may be provided in the outer shell in order to visually check the state in the incubator. Moreover, you may provide illumination in order to make it easy to visually observe the state in a culture bag inside an incubator.

The incubator is preferably provided with an air supply device that can be connected to an air supply pipe installed in the culture bag and can send gas into the culture bag.
It is preferable to install a heating device in the incubator in order to heat the inside of the culture bag and promote the culture. The heating device may be provided in either the outer shell or the culture bag, but is preferably provided in the outer shell in view of workability when assembling the incubator. The heating device may be provided either inside or outside the outer shell, but it is preferably provided inside from the viewpoint of heating efficiency. In the case of providing a warming device, even if it is partially provided, there is no problem because the internal temperature is made uniform by stirring, but it is preferable to provide it so as to cover the entire incubator. 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. When the outer shell is a metal can, an electromagnetic induction heating device can be used, and when the cylindrical container includes a resin layer having a polar group such as a carboxyl group, a high frequency induction heating device can also be used.
The incubator may be provided with a cooling device that can cool the inside of the culture bag in order to adjust the temperature in the culture bag or to terminate the production of substances and biological reactions. For example, a container that can accommodate an outer shell, and a container into which cooling water can flow between the container and the outer shell, may be installed so that the inside of the culture bag can be cooled.
The incubator is preferably provided with a pH adjusting device for maintaining or changing the pH of the culture solution in the culture bag. The pH adjusting device is preferably a device capable of supplying an acid or an alkali in order to adjust to a target pH.
The incubator is preferably provided with a control device such as a computer for controlling the heating device, the cooling device, the pH adjusting device and the like.

The incubator of the present invention can be used not only for culturing microorganisms but also for producing substances using microorganisms.
In addition, after culturing microorganisms and the like with the incubator of the present invention, the microorganisms and the like can be immobilized on a carrier in a culture bag and further used as a bioreactor. For example, after culturing microorganisms in a culture bag, an appropriate amount of a carrier for immobilizing microorganisms is added to the culture bag, the microorganisms are fixed while stirring with a stirrer, and further stirred in the incubator of the present invention via a communicating member. It is possible to perform the desired reaction by introducing aerobic conditions by introducing air or by introducing an inert gas such as nitrogen or argon. In this case, the carrier used is not particularly limited, but Kuraray manufactured by Kuraray Co., Ltd., APG manufactured by Nisshinbo Co., Ltd., or the like can be used.

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 a state in which a culture bag 10 according to an embodiment of the present invention is folded. FIG. 2 is a perspective view of the culture bag 10 according to one embodiment of the present invention. FIG. 3 is a perspective view of the outer shell 40. FIG. 4 is a perspective view of the incubator 100 according to one embodiment of the present invention, in which a part of the outer shell 40 is omitted for explanation.
The culture bag 10 of the present invention shown in FIG. 1 and FIG. 2 is used as a cylindrical container 11 with a bottom and lid by partially changing the specification of a 200 L drum inner bag Fuji liner 200 manufactured by Fujimori Industry Co., Ltd. A rotating device 22 made of PP is protruded from the vicinity of the center of the top surface 14 (lid) of the container through a bearing fixed with an adhesive, and a stirring device 20 having a stirring blade 21 made of PP in the cylindrical container 11 is provided. Airtightly stored. In order to store the stirring device 20 in the cylindrical container 11 in an airtight manner, the bottom surface 13 and the top surface 14 of the cylindrical container 11 may be sealed when heat-sealed, or may be enclosed in the center of the top surface of the cylindrical container 11. A spout similar to the spout 15 may be provided by a method to be described later and stored from here.

The body wall 12 of the cylindrical container 11 is obtained by cutting two PE inflation tubes having a diameter of 580 mm and a thickness of 100 μm into 870 mm and 1000 mm, and inserting the shorter tube into the longer tube. The reason why the trunk wall 12 has a double structure is to prevent the culture medium and the culture bag 10 from leaking even if a pinhole occurs in any of the trunk walls 12. Of course, a thick single layer tube may be used.
In this container, the lower ends of the two tubes that form the body wall 12 are aligned, a PE film having a diameter of 600 mm and a thickness of 100 μm is used as the bottom surface 13, and a PE film of the same type and the same material is formed 870 mm above the bottom surface 13. As shown in FIG. 1, the ring-shaped impulse sealer is used and heat-sealed with a width of 10 mm. The film to be the top surface 14 is previously provided with a hole having a diameter of 80 mm at the center and a position where the shortest distance between the circumference of the film and the circumference of the hole is 100 mm, and an inner diameter of 70 mm having a 20 mm wide flange. The PE outlet 15 is heat-sealed.

  An outer screw is provided on the cylindrical portion of the spout 15 and a cap 30 having an inner screw is screwed together. A hole is drilled in the center of the top surface of the cap 30 that seals the spout 15 at the center, and the rotating shaft 22 of the stirring device 20 accommodated from the spout 15 rotates through an airtight bearing. Inserted as possible. On the top surface of the outer (right side) cap 30, four holes are formed, and a communication member (not shown) consisting of four PE pipes whose outer ends are sealed is inserted into the cap 30 with an adhesive. Airtightly fixed. This communication member is connected to a tube made of a synthetic resin such as PE or PP, respectively, and a liquid supply pipe 32 for injecting a culture solution or the like into the cylindrical container 11, sampling, circulation of the culture solution or the like, It is used as a drain pipe 33 for discharging liquid that is no longer needed, an air supply pipe 34 for introducing a gas such as air or nitrogen, and an exhaust pipe 35 for discharging the gas. The drainage pipe 33 may be extended so that the opening end is positioned near the bottom surface of the cylindrical container 11. The air supply pipe 34 is preferably extended so that the open end is positioned near the bottom surface of the cylindrical container 11. In this case, if a weight is added to the distal end of the extending portion, the extending portion of the air supply pipe 34 sinks deeply into the culture solution, and the air supply pipe 34 stably hangs down. preferable. A plurality of holes may be opened along the extended portion of the air supply pipe 34. In this case, it is preferable that the end of the extending portion is sealed. The outer tips of the cylindrical containers of the liquid supply pipe 32 and the drainage pipe 33 are sealed by thermal fusion. Sealing may be performed by an appropriate means such as a pinch cock or a stopper. The ends of the air supply pipe 34 and the exhaust pipe 35 are sealed by attaching members each incorporating a HEPA filter as the filter 36 with an adhesive. The filter 36 may be directly heat-sealed with a nonwoven fabric or the like at the opening end.

A temperature sensor 16 using a thermocouple is attached to the outside of the body wall of the cylindrical container 11 with an adhesive tape. An adhesive may be used for pasting. Temperature information is sent from this sensor to a control device (not shown), and the heating temperature inside the culture bag 10 is controlled.
On the upper edge of the cylindrical container 11, one long tube forming the trunk wall 12 is left open. When the culture bag 10 is accommodated in the outer shell 40, the opening is folded back at the edge of the outer shell 40 and functions as a hanging portion 17 that suspends the culture bag 10 on the outer shell 40.
The cylindrical container 11 in which the stirring device 20 was accommodated was hermetically sealed with a cap 30 in which the rotating shaft 22 was inserted and a cap 30 having a communication member. In order to ensure airtightness, it is preferable to cover the lower end of the body portion of the cap 30 with an adhesive tape or a shrink tube. The cylindrical container 11 is sterilized with γ-rays, and the culture bag 10 of the present invention is obtained by sterilizing the entire interior including the stirring device 20. From an economic point of view, the γ-ray sterilization is preferably performed by folding the cylindrical container 11 as compactly as possible and collecting and packaging about 5 to 20 bags on an external cardboard or the like.

  The outer shell 40 shown in FIG. 3 has a 200 L stainless steel open drum can as an outer shell body 41. The inner diameter of the drum can (outer shell body) 41 is about 570 mm. A band-shaped heater 43 made of silicon rubber tarpaulin in which nichrome wire was knitted was disposed on the outer surface of the drum 41. A plurality of heaters 43 may be provided. The top plate 42 is provided with two holes through which the two members protruding from the culture bag 10, that is, the cap 30 into which the rotating shaft 22 is inserted and the cap 30 having the communication member can pass therethrough, and the spout 15 is protruded. It was. Further, the top plate 42 was provided with a window 47 having a diameter of 60 mm for internal observation, and a transparent acrylic plate was fixed with a bolt and sealed.

The hanging portion 17 of the culture bag 10 of the present invention was folded in the drum can 41 at the opening edge of the drum can 41 so as to cover the outer upper portion of the drum can 41. The top plate 42 was used as a canopy, and the drum 41 was tightened together with the hanging portion 17 of the culture bag 10 with an outer lever type band.
A metal motor fixing base 44 in which a cylindrical body having a flange was erected in the vicinity of the center of the top plate 42 was fixed with bolts. The rotating shaft 22 of the culture bag 10 is inserted into a metal pipe member (not shown) having a flange fixed to the motor 45 by a bolt so as to be coaxial with the rotating shaft of the metal pipe 46 and the motor 45 having flanges at both ends. Then, it was connected to the motor 45, and the flanges of the motor fixing base 44, the metal pipe 46, and the cylinder member of the motor 45 were fixed by the ferrule, respectively, to obtain the incubator 100 of the present invention shown in FIG.
When the inside of the culture bag does not become a positive pressure during the culture, for example, a plate having a U-shaped notch is sandwiched between the lower end of the cap 30 and the top plate 42 to connect the spout 15 to the top plate 42. It is preferable to fix to the hole.

A method of using the incubator 100 of the present invention will be described with reference to FIG.
A culture solution container in which a sterilized culture solution (not shown) is accommodated in the supply pipe 32 of the incubator 100 in which the culture bag 10 of the present invention is accommodated in the outer shell body 41 and covered with the top plate 42 is aseptically obtained. Connected. At the time of connection, aseptic joining apparatus SCD-IIB manufactured by Terumo Corporation was used. In addition, it can connect aseptically by performing a connection operation in an aseptic chamber, without using an aseptic joining apparatus.

The culture solution is filled in the culture bag 10 to which the culture solution container is aseptically connected through the supply pipe 32. Next, the culture is similarly injected from the supply pipe 32 into the culture bag 10 filled with the culture solution.
An air supply pipe 34 and an air supply tube are connected via a filter 36, and a gas such as air is pumped into the culture bag 10 as necessary using a pump or the like, and a motor is connected via a rotating shaft 22. At 45, the stirring blade 21 is rotated and the culture operation is performed while stirring.

The inside of the culture bag 10 is heated by a heater 43 provided on the outer surface of the outer shell 40 as necessary. The temperature is monitored by a temperature sensor 16 attached to the culture bag 10 via a gap in a hole through which the cap 30 of the top plate 42 passes. Based on the temperature information detected by the temperature sensor 16, the inside of the culture bag 10 is maintained at a predetermined temperature by the heater 43 by a control device (not shown). When the culture reaches a predetermined concentration, the culture is terminated, the culture bag 10 is opened, and the culture is recovered. The concentration of the culture is checked by aseptically connecting a suction tube from the drainage tube 33 into the culture bag 10 and sucking it with a pump or the like, and collecting a sample.
Next, when culturing, a new culture bag 10 is prepared, set in the outer shell 40, and cultured in the same manner.
The incubator 100 may be assembled after the culture bag 10 has been filled with the culture solution or the culture body, and finally the outer shell body 41 is covered with the top plate 42 to form the incubator 100.

Next, a second embodiment of the present invention will be specifically described with reference to the drawings.
FIG. 5 is a plan view of a state in which the culture bag 10 according to another embodiment of the present invention is folded. FIG. 6 is a schematic perspective view of an incubator 100 according to another embodiment of the present invention.
As in the first embodiment, the culture bag 10 of the present invention shown in FIG. 5 has a 200 L drum inner bag Fuji liner 200 manufactured by Fujimori Kogyo Co., Ltd. as a cylindrical container 11 with a bottom and a lid, and the specification is partially changed. It was.

  In the case of this embodiment, the stirring device 20 having the PP stirring blade 21 is housed in the cylindrical container 11 without airtightly protruding the PP rotating shaft 22 from the container top surface 14. A flange to which a magnetic material such as iron is fixed is joined to the upper end of the rotating shaft 22 accommodated in the culture bag 10. In the present embodiment, the iron plate is embedded in the flange.

The culture bag 10 has PE outlets 15 similar to those in the first embodiment, which are heat-sealed in the same manner as in the first embodiment, in a total of three places on the center and on the left and right sides thereof. Is screwed. Three communication members similar to those in the first embodiment are fixed to the right and left spout caps 30 in the same manner as in the first embodiment. The center cap 30 was left original without any holes.
Each spout 15 of the cylindrical container 11 in which the stirrer 20 is housed is hermetically sealed with a respective cap 30, and the entire interior including the stirrer 20 is made sterile as in the first embodiment. It was set as the culture bag 10 of invention.

  The obtained culture bag 10 was accommodated in a 200 L stainless steel open drum (outer shell 41) similar to the first embodiment. In the present embodiment, the suspended portion 17 of the culture bag 10 of the present invention is simply folded back at the edge of the drum can 41 so as to cover the outer upper portion of the drum can 41, and can be opened without being covered with a top plate. did. The culture bag suspension 17 may be fastened to the body of the drum 41 with a PP band or the like.

  Furthermore, the drum can 41 was accommodated in the lower half of the 250 L stainless steel open drum can (half-cut drum can 60) that was divided in two. Water is supplied from the water supply source 61 to the half drum 60, so that the inside of the culture bag 10 can be cooled as necessary, for example, when temperature adjustment or culturing is completed. The water supply is adjusted by an electronic valve 62, and the water used for cooling is sent to the drainage device 59. The band heater 43 is installed inside the drum 41 so as not to get wet.

  A stainless frame 63 was welded to the outer wall near the upper end of the half drum 60, and a motor 45 having a magnet at the tip of the rotating shaft was fixed in the middle of the horizontal frame of the frame 63 so that the rotating shaft would face downward. The tip of the rotating shaft is brought into close contact with the outside of the top surface of the cap 30 located at the center of the top surface of the culture bag 10, and the magnetic body of the stirring device 20 in the culture bag 10 is attracted by a magnetic force so It was made to adhere to. In this manner, the container top surface 14 of the culture bag 10 is fixed to the frame 63 via the cap 30 and the motor 45, and the respective rotation shafts of the motor 45 and the stirring device 20 slide on the top surface of the cap 30. The rotating force of the motor 45 can be transmitted to the rotating shaft 22 of the stirring device 20 while rotating.

The culture bag 10 is aseptically filled with a culture medium as a culture medium and a culture body as a culture target, and a tube is connected to one of the communicating members of the left cap 30 and a supply pipe 32 extending inside and outside the culture bag 10 did. From the liquid supply pipe 32, containers 56 and 57 each containing an acid such as hydrochloric acid for adjusting pH and an alkali such as sodium hydroxide, and a pump 55 that selects and feeds acid or alkali from these containers A pH adjusting device is connected.
Furthermore, a tube is aseptically connected to another communicating member of the left cap 30 to form an air supply pipe 34 extending in and out of the culture bag 10, and a compressor 58 for supplying gas is connected to this. In addition, the air supply pipe 34 has a loop shape in the vicinity of the tip where a large number of holes are formed in order to generate bubbles efficiently.

A tube is aseptically connected to one of the communicating members of the right cap 30 to form a drain tube 33 extending in and out of the culture bag 10, and the pH, temperature, dissolved oxygen of the culture solution in the culture bag 10 are added thereto. In addition, a culture solution measuring device for measuring the culture solution by circulating the culture solution is connected to sensors (pH / temperature sensor 51, dissolved oxygen concentration sensor 52, turbidity sensor 53) for measuring turbidity. In addition, the other communication member which is not illustrated is left with the outer end sealed as a spare.
In this way, the incubator 100 of the present invention shown in FIG. 6 was obtained.

  The method of using the incubator 100 is the same as that of the first embodiment, but the pump 55, the pH / temperature sensor 51, the dissolved oxygen concentration sensor 52, the turbidity sensor 53, and the electronic valve 62 are always used during the culture. It is measured and controlled by the control device 54. The broken-line arrows in FIG. 6 indicate that the control device 54 is a device that measures and controls.

Hereinafter, a culture example using the incubator 100 of the second embodiment will be described and further described.
The incubator 100 according to this embodiment is made of plastic in which holes 30 mm in diameter are formed in the caps 30, 30 attached to the left and right spouts 15, 15, and three communication members communicating in and out are formed in advance. The disc-shaped inner lid of each was inserted into each cap 30 and screwed tightly, and was sandwiched between the edge of the cap 30 and the tip of the spout 15 so that outside air would not enter the culture bag from other than the communicating member.
A silicone tube is connected to each communicating member, and a tube protruding outside the inner lid is clamped to allow access to the inside and outside as required. The tip of the tube is a plastic connector (not shown) instead of a filter. Was covered with a plastic bag and removed just before use,
The culture bag shown in FIG. 5 is sterilized in the same manner as the culture bag 10 described in the second embodiment, except that a fastener (not shown) is bonded to the bottom of the culture bag 10 and the air supply pipe 34 is fixed. 10 was produced.

  As in the second embodiment, each tube was connected under aseptic conditions to obtain the incubator 100 of the present invention shown in FIG.

The Escherichia coli HB101 strain was used as an indicator microorganism in a culture example using the incubator 100 of this embodiment. 10 ml of E. coli strain HB101 stored as a glycerol stock was seeded in 5 L of LB medium (tryptone 1%, yeast extract 0.5%, NaCl 1%), and cultured overnight by shaking to obtain a culture ( Pre-culture).
Similarly to the first embodiment, sterilized LB medium 150L is aseptically supplied from the supply pipe 32 to the culture bag 10 accommodated in the outer shell body 41 accommodated in the half drum 60, and further precultured. The whole culture was inoculated. The temperature of the culture solution was set to 37 ° C., the stirring blade 21 was rotated at 300 rpm, and at the same time, 200 L / min of air filtered through a filter (not shown) was supplied from the supply pipe 34 by the compressor 58, and the exhaust pipe Incubation was performed while maintaining the inside of the culture bag 10 at a positive pressure so that the outside air did not enter from 35.
When culturing, pH and temperature are monitored by the pH / temperature sensor 51 and controlled by the control device 54. If necessary, cooling water is supplied to the half drum 60 from the water supply source 61, or the outer shell body 41 The temperature of the culture was controlled by a heater 43 wound inside. Furthermore, the pump 55 was operated as needed, and acid or alkali was supplied from the acid container 56 and the alkali container 57.
After the start of the culture, the culture solution was refluxed using the drain tube 33, sampling was performed, and the turbidity sensor 53 was used to confirm the growth by obtaining measured values of turbidity (OD600) at 1 hour intervals. . OD600 means that the higher the value, the more microorganisms are growing.

On the other hand, as a comparative experiment, 100 ml of LB was dispensed into a 500 ml flask and a 500 ml baffle flask, 200 μl of each of the above precultured cultures was seeded, and shaking culture was performed while supplying sufficient air at 37 ° C. and 200 rpm. .
The results are shown in FIG.
As shown in FIG. 7, it can be seen that the culture bag of the present invention has a high proliferation of microorganisms as compared with the existing flask and further the baffle flask having a large oxygen uptake amount. From this result, it is expected that a higher yield 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, instead of the top plate, a motor may be fixed by passing a column or tube to the edge of the drum.
Further, instead of the stainless steel drum, a plastic drum or a fiber drum may be used, or a simple cylinder with no bottom and no lid may be used. They may be transparent.

  The present invention is applicable to culture bags and incubators that can perform a large-scale culture of cells and microorganisms and various operations on a large amount of microorganisms inexpensively and easily.

The top view of the state which folded the culture bag which concerns on the 1st embodiment of this invention. The perspective view of the culture bag which concerns on the 1st embodiment of this invention. The perspective view of the outer shell of the incubator concerning the 1st embodiment of the present invention. The perspective view of the incubator which concerns on the 1st embodiment of this invention. The top view of the state which folded the culture bag which concerns on the 2nd embodiment of this invention. The perspective view of the incubator which concerns on the 2nd embodiment of this invention. The figure which shows colon_bacillus | E._coli (HB101 stock | strain) growth curve when it culture | cultivates with the incubator of this invention, a 500 ml flask, and a 500 ml baffle flask.

Explanation of symbols

10. Culture bag 11. Cylindrical container 12. Container body wall 13. Container bottom 14. Container top (lid) 15. Spout 16. Temperature sensor 17. Hanging part 20. Stirring device 21. Stirring blade 22. Rotating shaft 30. Cap 32. Supply pipe 33. Drainage pipe 34. Air supply pipe 35. Exhaust pipe 36. Filter 40. Outer shell 41. Outer shell body (drum) 42. Outer shell top plate 43. Heater 44. Motor fixing base 45. Motor 46. Metal pipe 47. Observation window 51. pH / temperature sensor 52. Dissolved oxygen concentration sensor 53. Turbidity sensor 54. Control device 55. Pump 56. Acid container 57. Alkali container 58. Compressor 59. Drainage device 60. Half drum drum 61. Water supply source 62. Electronic valve 63. Frame 100. Incubator

Claims (15)

A culture bag in which a stirrer having a rotating shaft having a stirring blade is hermetically accommodated in a foldable bottomed and covered cylindrical container, and a hanging portion formed by an extension of a trunk wall is formed on the upper edge of the container A culture bag, which is provided over the entire circumference and is thermally welded with a spout that can be hermetically sealed with a cap on the lid of the container . The culture bag according to claim 1, wherein the spout has a plurality of heat-welded lids. The culture bag according to claim 1 or 2 , wherein the agitation device is stored in an airtight manner by projecting the rotating shaft from the spout, which is thermally welded to the lid of the container, so as to be rotatable outside the container. In the container, a tubular communication member that communicates the inside and outside of the container with one end projecting to the outside of the container is provided in a cap that hermetically seals the spout, and the container is hermetically disposed in the spout. The culture bag according to any one of claims 1 to 3, wherein the culture bag is provided. The culture bag according to any one of claims 1 to 4 , wherein the container includes a temperature sensor on an outer side of the trunk wall. The culture bag according to any one of claims 1 to 5 , wherein the inside of the culture bag is maintained in a sterile state. An incubator in which the culture bag according to any one of claims 1 to 6 is housed in a rigid outer shell. The incubator according to claim 7 , wherein the outer shell is a cylinder. The incubator according to claim 7 or 8 , wherein the incubator includes a driving device that rotates the rotating shaft. The incubator according to any one of claims 7 to 9 , wherein the incubator includes a heating device. The incubator according to any one of claims 7 to 10 , wherein the incubator includes a cooling device. The incubator according to any one of claims 7 to 11 , wherein the incubator includes a pH adjusting device. The incubator according to any one of claims 7 to 12 , wherein the incubator includes an air supply device. The incubator according to any one of claims 7 to 13 , wherein the incubator includes a sensor for measuring one or more of pH, temperature, dissolved oxygen and turbidity in the culture bag. The incubator according to claim 14 , further comprising a control device that controls one or more of pH, temperature, dissolved oxygen, and turbidity in the culture bag based on information from the sensor.

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