JP5570913B2 - Biological cell culture container and culture apparatus - Google Patents

Description

  The present invention relates to a culture vessel for culturing living cells and a culture apparatus equipped with the culture vessel.

  In the culture of living cells, it is desired to cultivate cheaply and safely in order to improve the productivity of the target product. The conventional method uses a culture tank made of stainless steel, and a variety of incidental facilities such as a steam supply device for sterilizing the inside of the culture tank and a cleaning means for cleaning the inside are essential. There was inevitably an increase in product costs. There is a method of using single-use containers as an effective culture technique for reducing the manufacturing cost. By making containers such as a culture tank and a medium tank into a single use, a sterilization system for sterilizing them and a cleaning system for performing cleaning are unnecessary, and production costs are expected to be reduced. Also, when changing the production object, there is no fear of contamination due to the previous product residue, and safety can be improved. Patent Document 1 describes a bioreactor including a device whose wall body is deformable. Patent Document 2 describes a culture apparatus provided with a means for determining and controlling a culture state from an image of cells in a culture bag. Patent Document 3 describes a shaking device for stirring a culture solution in a culture bag. Patent Document 4 describes a disposable bioreactor in which a sensor is arranged in a liquid transfer pipe connected to the bioreactor. Patent Document 5 describes a bioreactor including a ventilation sparger, a foam control means, a foldable bag container, and a support body that accommodates the bag body.

  These conventional techniques have been developed by applying techniques such as blood bags and infusion solution bags that have been used in the past to add functions essential for use as bioreactors. For this reason, the heavy use of single-use bioreactors has greatly contributed to cost reduction, but on the other hand, it has resulted in an increase in the workload of operators for preparations before use and removal after use. Yes. That is, for a single-use bioreactor, further improvement in usability and culture control is necessary.

  In particular, in the living cell culture process, it is necessary to monitor the dissolved oxygen concentration in the medium and the pH of the medium. However, for the single-use bioreactor described above, development of means for measuring dissolved oxygen concentration and pH in the medium with high accuracy, and development of means for attaching equipment for measuring dissolved oxygen concentration and pH in the medium, etc. Is not done enough.

Special Table 2004-535826 WO07-052716 WO07-052718 Special table 2009-536520 Special table 2009-539408

  Then, in view of the above-mentioned situation, the present invention aims to provide a culture vessel and a culture apparatus that can detect conditions such as dissolved oxygen concentration, dissolved carbon dioxide concentration and pH in a culture solution with high sensitivity. .

(1) A container main body, an agitating means that is disposed inside the container main body and stirs the culture solution stuck to the container main body, and an inner wall of the container main body. And a detection element for measuring the dissolved oxygen concentration or dissolved carbon dioxide concentration or pH of the culture solution embedded in the culture solution, and the detection element has a flow of the culture solution generated by the stirring means stirring the culture solution. A culture vessel characterized by being disposed at a position where it collides with the inner wall of the vessel body.
(2) The culture container according to (1), wherein the detection element is disposed at a position on the inner wall of the main body portion and facing the stirring means.
(3) The culture container according to (1), wherein the container main body is optically transparent at least at a portion where the detection element is disposed.
(4) The culture container according to (1), wherein the container main body is configured by a member that can be folded in a state in which an internal gas is exhausted.
(5) The culture container according to (1), further comprising a rigid top plate disposed on the upper surface of the container body.
(6) The stirring member includes a sheath member and a stirring blade disposed on the outer periphery of the sheath member, and the sheath member and the stirring blade according to the rotation of the rod-shaped member inserted into the sheath member. And the culture vessel according to (1), wherein
(7) It further comprises a rigid top plate disposed on the upper surface of the container main body, and a cylindrical member attached through the rigid top plate and the container main body, and one end of the sheath member is The culture vessel according to (6), which is attached to the cylindrical member.
(8) The stirring means includes a rotating shaft and a stirring blade disposed on an outer peripheral surface of the rotating shaft, and the rotating shaft is supported by a magnetic coupling member disposed at a base end portion of the rotating shaft. The culture vessel according to (1), which is rotatable.
(9) The culture vessel according to (8), wherein the rotating shaft is supported by at least a pair of sliding members.
(10) A rigid top plate disposed on the upper surface of the container main body, and a support member disposed on the rigid top plate and having a shape in a use state of the container main body ( 1) The culture vessel as described.
(11) The culture container according to any one of (1) to (10), a stirrer driving motor connected to the stirring means of the culture container and transmitting rotational force, a detection element of the culture container, and the culture container A light-receiving / light-emitting part disposed at a position facing through the container main body part, and a dissolved oxygen concentration in the culture solution based on fluorescence generated from the detection element of the culture container connected to the light-receiving / light-emitting part Or a culture device equipped with a measuring device for measuring dissolved carbon dioxide concentration or pH.
(12) The culture apparatus according to (11), wherein the measuring device is disposed in a positioning mechanism so as to be freely movable.
(13) Based on the dissolved oxygen concentration or dissolved carbon dioxide concentration or pH of the incubator measured by the measuring device, the supply amount of oxygen-containing gas or carbon dioxide to the culture vessel and the gas partial pressure or culture by the stirring means The culture apparatus according to (11), further comprising a control device for adjusting the liquid stirring conditions.

  In the culture container according to the present invention, it is possible to prevent the detection element attached inside from being contaminated, and to prevent a decrease in measurement accuracy even during long-term culture. Therefore, according to the culture container which concerns on this invention, a biological cell can be cultured efficiently on desired conditions.

  Similarly, since the culture apparatus according to the present invention is equipped with the culture container, it prevents contamination of the detection element attached to the inside of the culture container, and allows living cells to be collected over a long period of time without reducing measurement accuracy. It can be cultured.

It is a schematic block diagram which shows an example of the culture container to which this invention is applied, and a culture apparatus carrying this. It is a schematic block diagram of the stirring mechanism in the culture container to which this invention is applied, and the culture apparatus carrying this. It is a cross-sectional view of the rotating shaft which can be used as an example to the culture container to which this invention is applied, and the culture apparatus carrying this. It is a cross-sectional view of a rotating shaft that can be used as another example in a culture vessel to which the present invention is applied and a culture apparatus equipped with the same. It is a cross-sectional view of a rotating shaft that can be used as another example in a culture vessel to which the present invention is applied and a culture apparatus equipped with the same. It is a cross-sectional view of a rotating shaft that can be used as another example in a culture vessel to which the present invention is applied and a culture apparatus equipped with the same. It is a cross-sectional view of a rotating shaft that can be used as another example in a culture vessel to which the present invention is applied and a culture apparatus equipped with the same. It is a schematic block diagram which shows the other example of the culture container to which this invention is applied, and the culture apparatus carrying this.

Hereinafter, a culture container and a culture apparatus according to the present invention will be described in detail with reference to the drawings.
The culture container according to the present invention is for culturing various living cells while stirring the culture solution stuck inside. In particular, the culture container can be applied to culture of cells that produce substances that are main raw materials such as pharmaceuticals. In the present invention, examples of substances to be produced include proteins such as antibodies and enzymes, physiologically active substances such as low molecular compounds and high molecular compounds, and viruses. Examples of cells to be cultured include animal cells, plant cells, insect cells, bacteria, yeasts, fungi and algae. In particular, animal cells that produce proteins such as antibodies and enzymes are preferably cultured.

  For example, as shown in FIG. 1, the culture container according to the present invention is provided in the form of being mounted on a culture apparatus having various mechanisms for culturing living cells. Here, in the culture apparatus shown in FIG. 1, the culture container 1 is preferably a so-called disposable type (disposable type). In addition, although a disposable type culture container usually means what is discarded after one use (cultivation process), it also includes a case where it is used for two or more culture processes. That is, the disposable culture container is not limited to the number of times of use, and is synonymous with being able to be replaced with a different culture container.

  In this example, the culture container 1 includes a container main body portion made of a transparent synthetic resin film. For example, the container main body portion can be formed in a cylindrical shape having a diameter of 560 mm, a culture solution embedding height of 550 mm, and a culture volume of 100 L. In addition, the culture container 1 can be made into a use form by filling the inside with the sterilized gas. Moreover, the culture container 1 can be made into the state which the gas inside the container main-body part was substantially extracted and folded before use. That is, since the culture container 1 can be reduced in volume by making the gas inside the container main body portion almost exhausted, it has a shape suitable for storage and transportation.

  More specifically, as shown in FIG. 2, the culture container 1 includes a sheath member 31 inside the container main body, and stirring blades 2 a and 2 b attached at predetermined positions in the length direction of the sheath member 31. A detection element 3f attached to the inner wall of the container main body at a position facing the rotor 2b, a rigid top 6 attached to the outer upper surface of the container main body, and a support attached to the rigid top 6 It is comprised from the member 5, the rotation seal member 33 attached to the rigid top plate 6, and the cylindrical member 32 which was inserted in the rotation seal member 33 and to which the upper end part of the sheath member was attached.

  The detection element 3f includes, for example, a phosphor that generates fluorescence having different intensities depending on the dissolved oxygen concentration contained in the culture solution 7, the dissolved carbon dioxide concentration in the culture solution 7, or the pH of the culture solution 7, and the phosphor. And a film-like member having a function of blocking moisture and light and transmitting molecular oxygen. As such a detection element 3f, the product names of SP-PSt3-NAU-D5, SP-PSt3-YAU-D5, SP- manufactured by Taitec Co., Ltd. are used to detect the dissolved oxygen concentration contained in the culture solution 7. PSt6-NAU-D5 and SP-PSt6-YAU-D5 can be used, and the product name manufactured by Taitec Co., Ltd .: SP-CD1-D5-rMy- US can be used, and the product name: SP-HP5-D5 manufactured by Taitec Co., Ltd. can be used to detect the pH of the culture solution 7.

  The support member 5 is a plate-like member having a shape along the outer surface shape of the culture vessel 1 when the culture vessel 1 is filled with a gas and becomes usable. In addition, the support member 5 and the rigid top plate 6 may be damaged during the packing operation, the transport operation, the unpacking operation, and the unfolding operation with respect to the culture container 1 in a state where the internal gas is almost exhausted and folded. It functions to absorb and protect external forces.

  The rotary seal member 33 is attached to a substantially central portion of the rigid top 6 so as to penetrate the upper surface of the culture vessel 1. The rotary seal member 33 holds the cylindrical member 32 inserted therein in a rotatable and airtight manner. That is, the cylindrical member 32 has an outer diameter that is substantially the same as the inner diameter of the rotary seal member 33. Moreover, the sheath member 31 is airtightly attached to the end of the cylindrical member 32 facing the inside of the culture vessel 1. Further, the end of the sheath member 31 is sealed.

  The culture apparatus shown in FIG. 1 has a mechanism for mounting the culture container 1 configured as described above. That is, the culture apparatus includes a bottom surface support cover 16b that supports the bottom surface of the culture vessel 1, a side surface support cover 16s that supports the side surface of the culture vessel 1, and a side surface opening / closing device that supports the side surface of the culture vessel 1 and that can be opened and closed. And a support cover 16d. Although not shown, a concave groove for fitting the support member 5 of the culture vessel 1 is formed on the inner surface of the side support cover 16s in the height method. That is, when the culture vessel 1 is accommodated in the space formed by the bottom support cover 16b, the side support cover 16s, and the side opening / closing support cover 16d, the support member 5 of the culture vessel 1 is formed on the inner surface of the side support cover 16s. Fit into the recessed groove. The culture apparatus includes a gantry 19 for fixing the bottom surface support cover 16b, the side surface support cover 16s, and the side surface opening / closing support cover 16d.

  Further, the culture apparatus shown in FIG. 1 has various mechanisms for culturing living cells in the culture solution 7 stuck in the culture container 1 in a state where the culture container 1 configured as described above is mounted. It has. That is, the culture apparatus includes a light receiving / light emitting unit 3s attached to a position facing the detection element 3f disposed in the culture vessel 1, a dissolved oxygen concentration measuring device 3 connected to the light receiving / light emitting unit 3s, And a control device 4 for inputting a signal from the oxygen concentration measuring device 3. The light receiving / emitting unit 3s is attached at a position facing the detection element 3f disposed inside the culture vessel 1. FIG. 1 shows only the dissolved oxygen concentration measuring device 3 for measuring the dissolved oxygen concentration of the culture solution 7, but the culture apparatus is a dissolved carbon dioxide for measuring the dissolved carbon dioxide concentration of the culture solution. It has a concentration measuring device (not shown) and a pH measuring device (not shown) for measuring the pH of the culture solution. Here, the light receiving / light emitting unit 3s is not particularly limited as long as it can irradiate excitation light to the phosphor of the detection element 3f and can receive fluorescence from the phosphor. As the light receiving / emitting section 3s, for example, trade names: POF-L2.5-1SMA and POF-L2.5-2SMA manufactured by Taitec Corporation can be used. Further, the dissolved oxygen concentration measuring device 3 is not particularly limited as long as it measures the fluorescence received by the light receiving / emitting section 3s. As the dissolved oxygen concentration measuring device 3, for example, trade name: Fibox3 manufactured by Taitec Corporation can be used. Further, as a dissolved carbon dioxide concentration measuring device (not shown), a product name: pCO2-mini manufactured by Taitec Co., Ltd., and a product name: pH-1 mini manufactured by Taitec Co., Ltd. can be used as a pH measuring device.

  When the detection element 3f of the culture vessel 1 is in a position covered with the side support cover 16s with the culture vessel 1 attached, the side support cover 16s has an opening for allowing the light receiving / light emitting unit 3s to face the contents. Will have. The light receiving / light emitting unit 3s can be opposed to the detection element 3f of the culture vessel 1 by being attached to the opening. Moreover, it is preferable that the light receiving / light emitting unit 3 s is disposed by a positioning mechanism that can be relatively freely positioned with respect to the culture vessel 1. As the positioning mechanism, a slide mechanism that can freely slide in the circumferential direction and / or the height method along the outer periphery of the side support cover 16s can be employed. When the light receiving / light emitting unit 3s is freely positioned by the slide mechanism, the opening formed in the side support cover 16s is large enough to freely position the light receiving / light emitting unit 3s.

  As shown in FIG. 2, the culture apparatus includes a rod-shaped member 34 that is inserted into the sheath member 31 with the culture vessel 1 attached, a coupling 35 that transmits rotational force to the rod-shaped member 34, and a coupling 35. And a stirrer drive motor 11 that rotationally drives the rod-like member 34 via the shaft. In addition, the sheath member 31, the cylindrical member 32, and the rod-shaped member 34 are collectively shown as the rotating shaft 10 in FIG. The stirrer drive motor 11 is connected to the control device 4 and is driven and controlled.

  1 includes a first gas regulator 12 that supplies an oxygen-containing gas to the culture vessel 1, a second gas regulator 13 that supplies carbon dioxide to the culture vessel 1, and a culture vessel. 1 is provided with a third gas regulator 14 for supplying gas to the inside or discharging the gas inside. The first gas regulating device 12, the second gas regulating device 13, and the third gas regulating device 14 are respectively composed of a first gas passage tube 21, a second gas passage tube 22, and a gas filter 20 disposed in the middle. The culture vessel 1 is connected via a third gas flow path tube 23. Although not shown, the culture vessel 1 has a connection mechanism for hermetically connecting the first gas flow channel pipe 21, the second gas flow channel pipe 22, and the third gas flow channel pipe 23. The first gas channel tube 21 is connected to the air diffuser 17 for generating bubbles at the tip. The air diffusion means is not particularly limited, and any of known ring spargers, plastic porous materials, metal porous materials, inorganic porous materials, or a combination thereof is used. In addition, the size of the bubbles to be generated is appropriately selected according to the cells of the living body intended for culture. The second gas flow channel tube 22 and the third gas flow channel tube 23 are both connected to the culture vessel 1, and their tips are located in the upper space of the culture solution 7. Further, the first gas regulator 12, the second gas regulator 13, and the third gas regulator 14 are connected to the controller 4. Therefore, the control device 4 can control the timing and amount of gas supply from the first gas adjustment device 12, the second gas adjustment device 13, and the third gas adjustment device 14.

  Furthermore, the culture apparatus includes a liquid supply pipe 24 connected to a culture medium tank (not shown). The tip of the liquid supply tube 24 is located inside the culture vessel 1. The culture solution 7 is put into the culture vessel 1 from the culture solution tank via the liquid supply pipe 24.

  Furthermore, the culture apparatus includes a culture solution discharge pipe 25 connected to a tank (not shown). The culture solution discharge tube 25 is attached to the bottom surface of the culture vessel 1. The culture solution 7 stuck to the culture vessel 1 is discharged to a tank (not shown) through the culture solution discharge pipe 25.

  Furthermore, the culture device has a temperature adjusting means 15 connected to the control device 4. Although it does not specifically limit as a temperature control means, For example, the electric heater which heats the culture solution 7 can be used. The temperature control means 15 is disposed outside the side surface support cover 16s, for example. The heat supplied from the temperature control means 15 is transmitted to the culture solution 7 in the culture vessel 1 through the side support cover 16s. Moreover, it is preferable that the temperature control means 15 is provided with an overheat prevention control system so as not to exceed a preset temperature. Since the overheating prevention control system prevents heating to a predetermined temperature or higher, it is possible to prevent the culture vessel 1 from being damaged by heat.

  The operation | movement which attaches the culture container 1 is demonstrated about the culture apparatus comprised as mentioned above. First, in order to attach the culture vessel 1, the support member 5 of the culture vessel 1 is fitted to the concave groove formed on the inner surface of the side support cover 16s. Next, the rod-shaped member 34 is inserted from the cylindrical member 32 exposed above the culture apparatus 1 to the lower end portion of the sheath member 31, and the coupling 35 is fixed to the upper end portion of the cylindrical member 32 (see FIG. 2). At this time, the agitator drive motor 11 is fixed to the mount 19 by an attachment seat (not shown) whose attachment position can be freely changed, and is attached to the coupling 35 by adjusting the attachment seat. Thereby, the vibration accompanying the weight and rotation of the stirrer drive motor 11 can be supported by this mounting seat. That is, the culture vessel 1 can be released from the vibration accompanying the weight and rotation of the stirrer drive motor 11. Thereafter, the side opening / closing support cover 16d is closed and fixed.

  Next, the first gas channel tube 21, the second gas channel tube 22, the third gas channel tube 23, the liquid supply tube 24 and the culture solution discharge tube 25 are connected to the culture vessel 1. In this state, the sterilized gas from the third gas regulating device 14 is filled into the culture vessel 1 through the third gas flow channel pipe 23. As a result, the culture vessel 1 is expanded from the folded state to the usable state. The culture vessel 1 that has been deployed and is ready for use in this manner has an internal airtightness due to the configuration of the sheath member 31 whose lower end is sealed, the rotary seal member 33 to which the sheath member 31 is attached, and the like. Is held.

  In addition, when the culture vessel 1 is expanded from the folded state to the usable state as described above, the rigid top plate 6 made of a rigid member is airtightly installed on the portion corresponding to the ceiling of the culture vessel 1, Furthermore, since the support member 5 fixed to the rigid top plate 6 is provided, sufficient consideration is given so as not to damage the projections such as the stirring blades.

  When the light receiving / light emitting unit 3s is disposed in the positioning mechanism described above, the light receiving / light emitting unit 3s faces the detection element 3f of the culture container 1 after the culture vessel 1 is attached as described above. The position can be adjusted. For example, when the culture vessel 1 is formed of a synthetic resin film or the like, the culture vessel 1 is not always deployed in a certain shape and / or position depending on room temperature, humidity, gas amount to be filled, and the like. That is, the detection element 3f of the culture vessel 1 is not always at a fixed position. Therefore, by positioning the light receiving / light emitting portion 3s by the positioning mechanism, the detection element 3f and the light receiving / light emitting portion 3s can be more accurately opposed to each other, and measurement errors of dissolved oxygen concentration and pH can be prevented. Can do.

  As described above, the process for culturing cells using the culture apparatus with the culture vessel 1 attached will be described. First, a predetermined amount of the culture solution 7 is stuck inside the culture vessel 1 that maintains airtightness through the liquid supply pipe 24 attached to the culture vessel 1. At this time, since the internal pressure increases with the injection of the culture solution 7, the gas filled in the culture vessel 1 is discharged via the third gas flow channel pipe 23. In addition, during and after the completion of the culture solution, the third gas regulator 14 adjusts the pressure inside the culture vessel 1 to be slightly higher than the atmospheric pressure. Thereby, while being able to hold | maintain the shape of the culture container 1, the penetration | invasion of the microbe to the inside of the culture container 1 can be prevented.

  When the culture solution 7 is stuck in the culture vessel 1, the sheath member 31 of the portion immersed in the culture solution 7 is brought into close contact with the rod-shaped member 34 by pressure. Thereby, the sheath member 31 and the stirring blades 2a and 2b can rotate integrally with the rotation of the rod-shaped member 34. The cross-sectional shape of the rod-shaped member 34 is not particularly limited, but is preferably a non-circular shape. When the cross-sectional shape of the rod-shaped member 34 is circular, wrinkles and twists occur in the sheath member 31 that is in close contact with the rod-shaped member 34 due to the resistance applied to the stirring blades 2 a and 2 b that rotate with the rod-shaped member 34. There is a risk of damage from this part. On the other hand, when the cross-sectional shape of the rod-shaped member 34 is a non-circular shape, when the sheath member 31 comes into close contact with the outer circumferential surface of the rod-shaped member 34 due to the water pressure of the culture solution 7, On the other hand, a part will adhere relatively strongly. Specifically, the sheath member 31 is in close contact with the protruding portion in the cross section of the rod-shaped member 34. Thereby, even if the sheath member 31 is rotated together with the rod-shaped member 34, and resistance is loaded on the stirring blades 2a and 2b, this portion is caught so that the sheath member 31 does not wrinkle or twist, Breakage of the sheath member 31 can be prevented.

  Although it does not specifically limit as a non-circular cross-sectional shape, A shape as shown to FIGS. 3-7 can be illustrated. That is, examples include a substantially rectangular shape having at least one side as an arc as shown in FIG. 3, an elliptical shape as shown in FIG. 4, and a shape in which a part of a circle as shown in FIGS. it can. If the cross section of the rod-shaped member 34 has the shape shown in FIGS. 3 to 7, when the sheath member 31 comes into close contact with the outer circumferential surface of the rod-shaped member 34 due to the water pressure of the culture solution 7, It can be understood that some of them adhere relatively strongly. Therefore, by setting the cross section of the rod-shaped member 34 to the shape shown in FIGS. 3 to 7, it is possible to reliably avoid the inconvenience that the sheath member 31 is damaged by the rotation of the rod-shaped member 34.

  After or at the same time as the culture solution 7 is applied to the culture vessel 1 as described above, for example, biological cells are inoculated into the culture solution 7 via the liquid supply tube 24. Cell culture using the culture solution 7 is usually performed while rotating the stirring blades 2a and 2 at a predetermined rotational speed. The rotation speed of the stirring blades 2 a and 2 b is adjusted by controlling the rotation of the stirrer driving motor 11 by the control device 4. The rotation of the stirrer drive motor 11 is transmitted to the rotating shaft 10, and the culture solution 7 stuck in the culture vessel 1 is stirred and mixed by rotating the stirring blades 2 a and 2 b provided on the rotating shaft 10. Living cells that are fine particles are floating in the culture solution 7, and the culture solution 7 is caused to flow so that the cells do not deposit. In addition, when the cell of the living body to be cultured has anchorage dependency, a microcarrier that serves as a scaffold for proliferation is used. Living body cells having an anchorage dependency grow on the culture surface by contacting with the microcarriers. When the agitation speed is increased, mixing is strengthened, the amount of dissolved oxygen is increased, and the floating state of the cells in the living body is improved.

  Further, the temperature of the culture solution 7 at the time of cell culture is adjusted by the temperature adjusting means 15 so as to be a predetermined temperature. Further, at the time of cell culture, oxygen-containing gas is supplied to the culture solution 7 from the first gas regulator 12 through the first gas flow pipe 21 and the aeration means 17. Further, at the time of cell culture, carbon dioxide is supplied from the second gas regulating device 13 to the space above the culture solution 7 through the second gas passage tube 22. Thus, in the culture apparatus, the dissolved oxygen concentration in the culture solution 7 is controlled by increasing or decreasing the aeration gas amount and oxygen partial pressure of the oxygen-containing gas supplied from the first gas regulator 12. Further, in the culture apparatus, the pH in the culture solution 7 is controlled by increasing or decreasing the amount of carbon dioxide aeration gas and the partial pressure of carbon dioxide supplied from the second gas regulator 13.

  In particular, in the culture apparatus equipped with the culture vessel 1, the dissolved oxygen concentration in the culture solution 7 can be detected with high accuracy over a long period of time. The method for measuring the dissolved oxygen concentration will be described below, but the same applies to the method for measuring the dissolved carbon dioxide concentration and pH of the culture solution 7. In the culture apparatus, the detection element 3f attached to the inner wall surface of the culture vessel 1 and the light receiving / emitting unit 3s face each other with the culture vessel 1 mounted. When measuring the dissolved oxygen concentration of the culture solution 7, the excitation light having a predetermined wavelength emitted from the light emitting element of the light receiving / emitting unit 3s is transmitted through the wall surface of the culture vessel 1 and is provided on the detection element 3f. Irradiate. The phosphor of the detection element 3f is excited by excitation light and emits fluorescence when returning to the ground state. The fluorescence emitted from the phosphor is affected by the presence of oxygen, and the attenuation of fluorescence increases as the amount of dissolved oxygen increases. The fluorescence emitted from the detection element 3f is received by the light receiving / emitting section 3s, and the fluorescence intensity and its change with time are measured by the dissolved oxygen concentration measuring device 3. The excitation light irradiated from the light receiving / light emitting unit 3s and the fluorescence received by the light receiving / light emitting unit 3s are both transmitted through the wall surface of the culture vessel 1. Therefore, in the culture vessel 1, it is preferable that at least the region through which the excitation light and fluorescence are transmitted is transparent with respect to the wavelength region of the excitation light and fluorescence.

  The measured value of the dissolved oxygen concentration measured by the dissolved oxygen concentration measuring device 3 is output to the control means 4. For example, the control means 4 can control the dissolved oxygen concentration of the culture solution 7 to a desired value based on the input measured value of the dissolved oxygen concentration. Specifically, the control means 4 includes a method for controlling the stirrer drive motor 11 so as to increase or decrease the rotation speed of the stirring blades 2a and / or 2b, an aeration gas amount of oxygen-containing gas supplied from the first gas regulator 12, and / or The dissolved oxygen concentration can be controlled to be a predetermined value by either or both of the methods of controlling the first gas regulator 12 so as to regulate the oxygen partial pressure. In the control of the oxygen-containing gas by the first gas control device 12, the oxygen-containing gas is ventilated when the dissolved carbon dioxide concentration, which will be described later, is equal to or higher than a desired value and the reduction of the dissolved carbon dioxide concentration is controlled. The method by increasing or decreasing the gas amount is prioritized, and when the dissolved carbon dioxide concentration is not more than a desired value and the reduction of the dissolved carbon dioxide concentration is not controlled, the method of adjusting the oxygen partial pressure of the oxygen-containing gas is prioritized.

  The measured value of the dissolved carbon dioxide concentration measured by a dissolved carbon dioxide concentration measuring device (not shown) is output to the control means 4. For example, the control means 4 can control the dissolved carbon dioxide concentration of the culture solution 7 to be a desired value or less based on the input measurement value of the dissolved carbon dioxide concentration. Specifically, the control means 4 is a method for controlling the stirrer drive motor 11 to increase the rotation speed of the stirring blades 2a and / or 2b when the dissolved carbon dioxide concentration exceeds a desired value, the first gas adjustment The dissolved dissolved carbon dioxide concentration becomes a predetermined value by either or both of the methods of controlling the first gas control device 12 so as to adjust the gas flow rate and / or the oxygen partial pressure of the oxygen-containing gas supplied from the device 12. Can be controlled. Specifically, when the dissolved carbon dioxide concentration exceeds a desired value, a method for increasing the rotational speed of the stirring blades 2a and / or 2b, a method for increasing the amount of aeration gas of the oxygen-containing gas to be supplied, and a method for supplying The dissolved dissolved carbon dioxide concentration is controlled to be a predetermined value by appropriately using a method for reducing the oxygen partial pressure of the oxygen-containing gas.

  The measured pH value measured by a pH meter (not shown) is output to the control means 4. For example, the control means 4 can control the pH of the culture solution 7 to a desired value based on the input measured value of pH. Specifically, when the pH exceeds a desired value, the control means 4 controls the second gas regulator 13 to increase the amount of carbon dioxide aeration gas supplied from the second gas regulator 13. When the pH is lower than a desired value, the second gas regulator 13 is controlled to reduce the amount of carbon dioxide aeration gas supplied from the second gas regulator 13 so that the carbon dioxide aeration gas amount is zero. Even if the pH is further lowered, the neutralizer addition device (not shown) is controlled so as to add an alkaline neutralizer.

  At this time, in the culture apparatus according to the present invention, since the detection element 3f disposed in the culture vessel 1 is positioned so as to face the stirring blade 2b, the culture medium in the circumferential direction generated by the rotation of the stirring blade 2b. 7 flows strongly against the surface of the detection element 3f. The flow of the culture solution 7 can prevent various components (live cells, dead cells, cellular acidic substances, medium components, etc.) from adhering to the surface of the detection element 3f. In particular, when living cells contained in the culture solution 7 adhere to the detection element 3f, the living cell confidence consumes oxygen, so the dissolved oxygen concentration cannot be measured accurately. In particular, in the culture device according to the present invention, it is possible to always prevent various components from adhering to the detection element 3f even when cells are cultured for a long period of time. The dissolved oxygen concentration can be measured.

  In the present invention, when the detection element 3f disposed in the culture vessel 1 is positioned so as to face the stirring blade 2b, the circumferential flow of the culture solution 7 generated by the rotation of the stirring blade 2b is as described above. It means a position where it can act, and does not mean that the height position of the stirring blade 2b and the height position of the detection element 3f are exactly the same. For example, even if the height position of the detection element 3f is deviated from the height position of the stirring blade 2b, the detection element 3f faces the stirring blade 2b as long as the above-described culture fluid flow acts.

  As described above, according to the culture apparatus equipped with the culture vessel 1 according to the present invention, it is possible to perform good culture control even in long-term culture by preventing a decrease in measurement accuracy due to contamination on the surface of the detection element 3f. Furthermore, since the dissolved oxygen concentration, dissolved carbon dioxide concentration, and pH of the culture solution 7 can be measured by the configuration including the light receiving / emitting portion 3s at the position facing the detection element 3f through the outer wall surface of the culture vessel 1, the use of an optical fiber is possible. High-sensitivity measurement can be maintained without causing a cost increase due to, and a decrease in sensitivity due to light attenuation and stray light penetration. By providing the culture vessel 1 and the culture apparatus having these effects, the culture can be continued for a long period of time, and the productivity of the target substance and the safety of the product can be improved.

  After the above-described cell culture is completed, the culture solution 7 can be discharged from the culture solution discharge pipe 25 and stored in a tank (not shown). During the above-described cell culture, the liquid supply tube 24 and the culture solution discharge tube 25 maintain a sealed state to prevent invasion of microorganisms and leakage of the internal solution.

  It should be understood that the culture apparatus equipped with the culture vessel 1 shown in FIGS. 1 and 2 may have a configuration not shown. The culture apparatus on which the culture vessel 1 is mounted may include, for example, a plurality of pipes for sampling, pH adjustment drug injection, medium exchange, and the like. In addition, the culture apparatus equipped with the culture vessel 1 may be provided with a liquid inlet / outlet nozzle used when a desired reagent is added to a part of the culture solution 7 in the culture vessel 1. Furthermore, the culture apparatus in which the culture vessel 1 is mounted may include a gas supply facility such as air, oxygen, nitrogen, and carbon dioxide, a hot / cold water supply facility, and a water supply / drainage facility.

  On the other hand, in the culture apparatus equipped with the culture vessel 1 shown in FIGS. 1 and 2, the rod member 34 is inserted into the sheath member 31 of the culture vessel 1, and the sheath member 31 is brought into close contact with the rod member 34 by the water pressure of the culture solution 7. Thus, the rod-shaped member 34 and the sheath member 31 are integrally driven to rotate. However, the present invention is not limited to such a configuration. For example, as illustrated in FIG. 8, a configuration in which the rotation shaft 10 of the culture vessel 1 is rotationally driven using a so-called magnet coupling may be used.

  Specifically, as shown in FIG. 8, the culture vessel 1 includes a rigid rotating shaft 10 to which stirring blades 2 a and 2 b are attached, and a first magnetic coupling connected to one end side of the rotating shaft 10. A member 40; a first sliding member 41 into which one end of the rotating shaft 10 is inserted; an agitator support member 42 fixed to the rigid top plate 6; and a second sliding member attached to the agitator support member 42. 43. In the culture vessel 1, the rotating shaft 10 is supported by being inserted into the first sliding member 41 and the second sliding member 43 that are arranged at a predetermined interval. That is, the contact point between the first sliding member 41 and the rotary shaft 10 is an upper fulcrum of the rotary shaft 10, and the contact point between the second sliding member 43 and the rotary shaft 10 is a lower fulcrum of the rotary shaft 10. The first sliding member 41 is fixed to the rigid top plate 6, and the second sliding member 43 is fixed to the rigid top plate 6 via the stirrer support member 42.

  On the other hand, the culture apparatus to which the culture vessel 1 is attached includes a stirrer drive motor 11, a second magnetic coupling member 44 attached to the end of the drive shaft 46 of the stirrer drive motor 11, and a second magnetic coupling member 44. An attachment guide 45 to be fixed is provided.

  In the culture device configured as described above, the rotation of the drive shaft 46 of the stirrer drive motor 11 is transmitted to the rotary shaft 10 by the magnetic force between the first magnetic coupling member 40 and the second magnetic coupling member 44. The Rukoto. Thereby, similarly to the culture apparatus shown in FIGS. 1 and 2, the stirring blades 2 a and 2 b can be rotated in the culture solution 7 even in the culture apparatus having the configuration shown in FIG. 8.

  In particular, in the culture apparatus shown in FIG. 8, it is not necessary to provide a through hole for mechanical sealing in the rigid top plate 6, and the airtightness of the culture vessel 1 can be further increased. Therefore, in particular, in the culture apparatus shown in FIG. 8, the possibility of microbial contamination can be further reduced, and the safety of the product can be improved. Furthermore, in the culture apparatus shown in FIG. 8, the culture vessel can be attached and detached more easily, and the working efficiency can be improved.

DESCRIPTION OF SYMBOLS 1 ... Culture container, 2a, 2b ... Stirring blade, 3f ... Detection element, 3s ... Light-receiving / light emission part, 3 ... Dissolved oxygen concentration measuring device, 4 ... Control apparatus, 5 ... Support member, 6 ... Rigid top plate, 10 ... Rotating shaft, 11 ... Stirrer drive motor, 12 ... First gas regulator, 13 ... Second gas regulator, 14 ... Third gas regulator, 16 ... Support cover, 19 ... Base, 21 ... First gas channel tube 22 ... second gas channel tube, 23 ... third gas channel tube, 24 ... liquid supply tube, 25 ... culture medium discharge tube, 31 ... sheath member, 32 ... cylindrical member, 34 ... bar-like member, 40 ... first 1 magnetic coupling member, 44 ... 2nd magnetic coupling member

Claims (10)

A container body,
An agitation means for agitating the culture solution disposed in the container body part and stuck to the container body part;
A detector for measuring the dissolved oxygen concentration or dissolved carbon dioxide concentration or pH of the culture solution disposed on the inner wall of the container main body and stretched on the container main body;
The stirring means includes a sheath member and a plurality of stirrer blades disposed on the outer periphery of the sheath member, and the sheath member and the stirring blade are rotated along with the rotation of the rod-shaped member inserted into the sheath member. And rotate,
The rod-shaped member has a non-circular cross section,
The sheath member and the stirring blade are rotated with the rotation of the rod-shaped member by closely contacting the rod-shaped member by the water pressure of the culture solution stuck to the container main body portion,
The culture container, wherein the detection element is disposed at a position where a culture fluid flow generated by the stirring means stirring the culture fluid acts on the inner wall of the container body.   2. The culture container according to claim 1, wherein the detection element is disposed at a position on the inner wall of the container main body and facing the stirring means.   2. The culture container according to claim 1, wherein at least a portion where the detection element is disposed is optically transparent in the container main body.   2. The culture container according to claim 1, wherein the container main body is constituted by a member that can be folded in a state in which an internal gas is exhausted.   The culture vessel according to claim 1, further comprising a rigid top plate disposed on the upper surface of the vessel main body.   A rigid top plate disposed on the upper surface of the container main body, and a cylindrical member attached through the rigid top plate and the container main body, wherein one end of the sheath member is the cylindrical member The culture container according to claim 1, wherein the culture container is attached to the culture container.   2. A rigid top plate disposed on an upper surface of the container main body, and a support member disposed on the rigid top plate and having a shape in a use state of the container main body. Culture vessels. A culture vessel according to any one of claims 1 to 7;
An agitator drive motor connected to the agitation means of the culture vessel and transmitting rotational force;
A light receiving / emitting section disposed at a position facing the detection element of the culture container and the container main body of the culture container;
A culture apparatus comprising: a measuring device connected to the light receiving / emitting unit and measuring a dissolved oxygen concentration or a dissolved carbon dioxide concentration or pH in a culture solution based on fluorescence generated from a detection element of the culture vessel.   9. The culture apparatus according to claim 8, wherein the measuring device is disposed in a positioning mechanism so as to be movable in a positionable manner.   Based on the dissolved oxygen concentration or dissolved carbon dioxide concentration or pH in the culture solution measured with the measuring device, the supply amount and gas partial pressure of the oxygen-containing gas or carbon dioxide to the culture vessel or the culture solution by the stirring means 9. The culture apparatus according to claim 8, further comprising a control device for adjusting the stirring conditions.

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