WO2017026199A1 - シングルユース細胞培養装置および培養バッグ - Google Patents
シングルユース細胞培養装置および培養バッグ Download PDFInfo
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- WO2017026199A1 WO2017026199A1 PCT/JP2016/069631 JP2016069631W WO2017026199A1 WO 2017026199 A1 WO2017026199 A1 WO 2017026199A1 JP 2016069631 W JP2016069631 W JP 2016069631W WO 2017026199 A1 WO2017026199 A1 WO 2017026199A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/14—Bags
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
- C12M1/06—Apparatus for enzymology or microbiology with gas introduction means with agitator, e.g. impeller
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/007—Flexible bags or containers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/26—Constructional details, e.g. recesses, hinges flexible
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/48—Holding appliances; Racks; Supports
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/18—Flow directing inserts
- C12M27/20—Baffles; Ribs; Ribbons; Auger vanes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
Definitions
- the present invention relates to a single-use cell culture device and a culture bag.
- a culture method for culturing living cells such as microorganisms, animal cells, plant cells, and fungi is used.
- living cell culture techniques stainless steel culture tanks have been used. Living cells and culture solution are accommodated in the culture tank, and the culture solution is stirred by the stirring means.
- the culture tank and the stirrer are washed and sterilized before storing the culture solution in the culture tank.
- an auxiliary facility such as a steam supply device for sterilizing the inside of the culture tank and a cleaning mechanism for cleaning is required, and the operation is not easy.
- a single-use culture system for culturing living cells using a disposable flexible bag that is, a single-use culture system
- a culture system includes a flexible single-use culture bag made of a plastic film and the like, and a support (housing portion) that holds the culture bag and holds the shape. ing.
- Patent Document 1 discloses a culture bag that contains a culture solution containing a culture medium and cultures the culture medium, and a bag body formed of a plastic material and freely rotatable in the bag body.
- a culture bag is described in which baffles are provided with baffle holes through which the culture object can pass.
- this patent document 1 describes that the baffle of the culture bag is heat-sealed together with the film constituting the culture bag and integrally joined. Further, as another aspect, it is described that the baffle in the culture bag is held by a baffle holding part provided outside the bag body.
- Patent Document 2 also discloses a stirred tank reactor system, which is: (i) a flexible bag having at least one opening, the bag serving as a sterilization container for a fluid medium (Ii) a shaft disposed within the bag; (iii) an impeller attachable to the shaft, wherein the impeller agitates the fluid medium to provide a hydrodynamic environment
- a stirred tank reactor system is described that includes an impeller used to: and (iv) a bearing attached to the shaft and the opening of the bag.
- the housing includes a plurality of baffles, and the bag is folded around the baffles.
- the baffle described in Patent Document 1 is provided in order to promote the flow in the vertical direction in the culture solution and improve the stirring efficiency.
- the joining is performed when pressure is applied to the joining portion due to fluid force applied to the baffle.
- the culture medium may leak due to tearing or peeling off of the portion.
- the culture solution may leak from a place where the joining is incomplete.
- the culture bag may be torn and the culture fluid may leak when pressure is applied to the portion held by the baffle holding portion.
- the present invention has been made in view of the above circumstances, and can provide a single-use cell culture device and a culture bag that can improve the stirring efficiency of the culture solution and further reduce the fear of leakage of the culture solution. Let it be an issue.
- the single-use cell culture apparatus that has solved the above-described problems includes a housing part that houses a culture bag that has flexibility and can contain a culture solution therein.
- a protruding portion having a curved convex shape is provided on a part of the contact surface that contacts the bag.
- the culture bag according to the present invention is housed in a housing part of a single-use cell culture device, has flexibility, can enclose a culture solution therein, and is provided in a curved portion provided in a part of the housing part.
- a concave portion having a shape corresponding to the convex protrusion is provided.
- the present invention it is possible to provide a single-use cell culture device and a culture bag that can improve the stirring efficiency of the culture solution and further reduce the concern of leakage of the culture solution.
- FIG. 1 is a schematic configuration diagram illustrating one embodiment of a single-use cell culture device according to the present invention. It is a notch figure of the housing part explaining the one aspect
- FIG. 3 is a sectional view taken along line III-III in FIG. 1. It is sectional drawing explaining the other aspect of a projection part. It is sectional drawing explaining the other aspect of a projection part. It is sectional drawing explaining the other aspect of a projection part. It is a notch figure of the housing part explaining the other aspect of a projection part. It is a notch figure of the housing part explaining the other aspect of a projection part. It is a notch figure of the housing part explaining the other aspect of a projection part. It is a notch figure which shows the other aspect of a housing part and a protrusion.
- cell culture device single-use cell culture device
- culture bag according to the present invention
- the cell culture device and culture bag according to the present invention can be applied when culturing microorganisms or animal and plant cells that produce substances as main raw materials such as pharmaceuticals and health foods.
- the substance to be produced is not limited in any way, and examples thereof include proteins such as antibodies and enzymes, physiologically active substances such as low molecular compounds and high molecular compounds, and viruses.
- carotenoids such as ⁇ -carotene and astaxanthin
- pigments such as chlorophyll and bacteriochlorophyll
- phycobilin proteins such as phycocyanin used for coloring foods and cosmetics
- physiologically active substances such as fatty acids.
- cells to be cultured include animal cells, plant cells, photosynthetic bacteria, microalgae, cyanobacteria, insect cells, bacteria, yeasts, fungi and algae. In particular, it is preferable to culture animal cells that produce proteins such as antibodies and enzymes.
- the medium used for the culture is not particularly limited, and any conventional medium can be used.
- FIG. 1 is a schematic configuration diagram illustrating one embodiment of a cell culture device according to the present invention.
- the cell culture device 100 includes a housing part 2 that houses a culture bag 1.
- the housing portion 2 is provided with curved convex protrusions 4 and 4 on a part of the abutting surface that abuts the culture bag 1.
- FIG. 1 illustrates a state in which the culture bag 1 in which the culture solution 7 is enclosed is housed in the housing part 2.
- the cell culture device 100 includes a stirring means for stirring the culture solution 7 stored in the culture bag 1.
- a stirring drive device 5 fixed to the support frame 3 is provided below the housing portion 2 as the stirring means.
- the agitation drive device 5 agitates the culture solution 7 contained in the culture bag 1 by rotating the housing part 2 eccentrically from the central axis.
- the cell culture device 100 includes a gas supply facility such as air, oxygen, nitrogen and carbon dioxide, a hot / cold water supply facility, and a water supply / drainage facility that are indispensable for the culture facility. is doing.
- the cell culture apparatus 100 includes a medium whose composition has been prepared in advance for culturing target cells, a supply facility for an additional medium to be added during culturing, and the like.
- Both the housing part 2 and the support frame 3 need only be strong enough to support the weight and pressure of the culture bag 1 filled with the culture solution 7 and maintain the shape, such as metal or hard plastic. It doesn't matter.
- FIG. 2 is a cut-away view of the housing portion 2 for explaining one aspect of the protruding portion 4 shown in FIG. 3 is a cross-sectional view taken along the line III-III in FIG.
- the culture bag 1 is shown floating from the inner wall of the housing part 2, but actually, the inner wall surface of the housing part 2 is caused by the pressure of the enclosed culture solution 7.
- 4A to 4C are cross-sectional views for explaining other aspects of the protrusion 4 respectively.
- the protrusion 4 can have a substantially triangular prism shape, but is not limited to this, and can have a substantially semi-cylindrical shape as shown in FIG. As shown to 4B, it can be set as the substantially triangular prism shape which the two surfaces contact
- the protruding portion 4 in the present invention only needs to be formed with a slightly rounded (curved) portion 4a as a side so as not to be broken when pressure is applied to the culture bag 1.
- the cell culture device 100 can promote the flow in the vertical direction when the stirred culture solution 7 hits the protruding portion 4. Therefore, the cell culture device 100 can improve the stirring efficiency of the culture solution 7.
- both the upper end part 4b and the lower end part 4c of the projection part 4 shown in FIG. 2 can be made into an open state, it can also be made into a closed state by closing an opening part using a board
- the side 4d forming the upper end portion 4b and the lower end portion 4c is preferably processed so as to be rounded. In this way, it is possible to make the culture bag 1 more difficult to tear.
- the roundness of the side portion 4a is, for example, such that the radius of curvature is 0.05 to 0.5 times the length of the bottom side 4e. If the curvature radius of the part 4a used as a side is made into this range, since the part 4a used as a side is moderately rounded, the culture bag 1 can be made difficult to tear. From the viewpoint of making the culture bag 1 more difficult to break, the radius of curvature of the side portion 4a is preferably 0.2 or more, and more preferably 0.45 or less.
- the height W of the protrusion 4 is preferably in the range of 0.05 to 0.15 in terms of W / D with respect to the inner dimension D of the housing 2.
- the height W of the protrusion 4 is preferably 0.08 or more in terms of W / D, and more preferably 0.13 or less.
- the height W of the protrusion 4 can be 0.5 to 5 times the length B of the bottom in the cross section of the protrusion 4.
- the culture medium is not disturbed excessively as described above without excessively disturbing the swirling flow of the culture liquid 7 being stirred. 7 can be further encouraged to flow in the vertical direction. Therefore, the cell culture device 100 can further improve the stirring efficiency.
- the height W of the protrusion 4 is preferably 1 or more, and preferably 3 or less, of the length B of the bottom of the protrusion 4.
- the length L of the protrusion 4 in the longitudinal direction is 0.05 to 1 in terms of L / H with respect to the liquid level height H of the culture solution 7 sealed in the culture bag 1. It is preferable that it exists in the range.
- the protrusion 4 may be divided into a plurality of parts on the same line, and the length L in this case is a plurality of protrusions 4 arranged on the same line. It is assumed that the lengths 4 and 4 are totaled (in this example, the distance between the protrusion 4 and the protrusion 4 is not included in the length L).
- FIG. 5 is a cut-away view of the housing portion 2 for explaining another aspect of the protruding portion 4.
- the cell culture device 100 can further improve the stirring efficiency.
- the length L in the longitudinal direction of the protrusions 4 is preferably 0.3 or more in terms of L / H, and preferably 0.8 or less.
- the protruding portion 4 can be provided in a direction parallel to the vertical direction on the side wall portion of the housing portion 2. Further, as shown in FIG. 6, the protruding portion 4 can be provided on the side wall portion of the housing portion 2 at a predetermined angle ⁇ with respect to the vertical direction.
- FIG. 6 is a cutaway view of the housing portion 2 for explaining another aspect of the protruding portion 4.
- the predetermined angle ⁇ can be set arbitrarily.
- the predetermined angle ⁇ is preferably more than 0 ° and 45 ° or less. Even when tilted in this way, the flow in the vertical direction can be promoted to the culture solution 7 as described above.
- FIG. 7 is a cutaway view showing another embodiment of the housing part 2 and the protrusion part 4.
- a fixing mechanism 21 having one or more holes 21 a in the housing portion 2 is provided.
- fixed part 20 for fixing to the hole 21a of the fixing mechanism 21 is provided in the projection part 4.
- the fixing mechanism 21 may be anything as long as the protruding portion 4 can be fixed by the fixing portion 20, but can be formed of, for example, a punching metal or a metal mesh.
- the fixing portion 20 is preferably provided on the opposite side of the portion 4 a serving as the side, that is, on the bottom side of the protrusion 4.
- the fixing part 20 may be, for example, a convex body having a shape that fits a hole in a punching metal or a hook that is engaged with a mesh of a wire mesh.
- the culture bag 1 As shown in FIG. 1, the culture bag 1 according to the present invention is particularly limited as long as it is housed in the housing part 2 of the cell culture device 100, has flexibility, and can enclose the culture solution 7 therein. It can be used without being done.
- the culture bag 1 for example, a bag made of a multilayer film of ethylene / vinyl / acetate or ethyl / vinyl / alcohol can be suitably used.
- single use bags for pharmaceutical packaging are commercially available from various companies, and can be arbitrarily selected and used.
- commercially available culture bags are generally sterilized in advance by gamma rays, ultraviolet rays, ethylene oxide gas, etc. to maintain sterility, and the contents such as gas and liquid are almost removed and provided in a folded state. Has been.
- a concave portion 1a (see FIG. 3) having a shape corresponding to the curved convex protrusion 4 provided in a part of the housing portion 2.
- the protrusion 4 of the housing part 2 and the concave part 1 a of the culture bag 1 coincide with each other. That is, since an extra pressure is not applied to the culture bag 1, it is possible to further prevent the culture bag 1 from being torn.
- the culture bag 1 is provided with an in-liquid aeration gas supply pipe 8, a gas phase gas supply pipe 9, a culture medium supply pipe 10, a culture liquid discharge pipe 11, and a gas discharge pipe 12.
- An aeration means 13 for generating bubbles is connected to the tip of the submerged aeration gas supply pipe 8 provided in the culture bag 1.
- the aeration means 13 is provided at the bottom along the side wall in the culture bag 1 by fusion or the like.
- a valve 6 is provided in each of the gas supply pipe 8 for in-liquid aeration, the gas supply pipe 9 for the gas phase, the medium supply pipe 10, the culture medium discharge pipe 11, and the gas discharge pipe 12, so ON / OFF and the flow rate thereof can be appropriately controlled.
- the submerged gas supply pipe 8, gas phase gas supply pipe 9, and gas discharge pipe 12 are provided with a gas filter 14 for preventing invasion of microorganisms from the outside.
- sampling, injection of a drug for pH adjustment, a medium replacement pipe, and the like can be provided, but the description in FIG. 1 is omitted.
- a culture state measurement means a device for measuring pH, temperature, dissolved oxygen concentration, dissolved carbon dioxide concentration, and the like and respective sensors are provided. Described as 15.
- the culture bag 1 is provided in a folded state, and is positioned and installed so as to be in a predetermined mounting arrangement of the housing part 2 by the position fixing supports 16a and 16b.
- the culture bag 1 can be inserted and arranged from the opening part in the upper part, but the opening part is provided in the side wall part. Can be inserted from the side wall.
- the culture solution 7 in which the target cells and the medium are suspended is filled into the culture bag 1 through the medium supply tube 10.
- the culture solution 7 may be filled after being prepared in advance to have a predetermined cell concentration, or may be prepared to have a predetermined cell concentration by seeding the cells after filling the medium.
- the culture medium supply tank or culture solution preparation tank and the culture medium supply pipe 10 which are not shown in the figure are connected aseptically.
- the culture bag 1 is pressure-bonded to the inner wall surface of the housing part 2 or the protrusion part 4 by the pressure of the filled culture solution 7 due to gravity.
- a baffle (baffle plate) is formed on the inner surface of the culture bag 1 in contact with the housing portion 2 by the curved convex protrusion 4.
- the support base 3 is provided with the agitation drive device 5 for the culture solution 7 by a shaking method by rotating the culture bag 1 and the housing portion 2 together with the culture bag 1 eccentric from the central axis.
- the stirring drive device 5 By driving the stirring drive device 5, the culture solution 7 in the culture bag 1 can be fluidized and stirred.
- the agitation driving device 5 causes the culture solution 7 to generate a swirling flow in a substantially horizontal direction.
- the projections 4 are illustrated).
- the direction of the swirl flow is turned to generate a vertical flow, and the culture solution 7 can be mixed efficiently so that it becomes uniform.
- the culture bag 1 is connected to the gas supply means 17 for submerged gas via the gas supply pipe 8 for submerged gas, and is connected to the gas supply means 18 for gas phase via the gas supply pipe 9 for gas phase. It is connected.
- the gas supply means 17 for aeration in the liquid and the gas supply means 18 for the gas phase are driven to ventilate a predetermined concentration of oxygen mixed gas into the culture bag 1 and start the culture.
- Oxygen gas vented from the liquid surface or in the liquid dissolves in the liquid, and as described above, the direction of the swirl flow is turned and flows in the culture bag 1 while generating a vertical flow. In the meantime, oxygen can be uniformly supplied to the cells in the culture solution 7, and the stirring efficiency of the culture solution can be improved.
- the pH, temperature, DO (dissolved oxygen concentration), and DCO 2 (dissolved carbon dioxide concentration) of the culture solution 7 are measured by a corresponding sensor and measuring instrument typically described as the measuring means 15 and input to the control device 19.
- the control device 19 maintains the predetermined pH, DO, and DCO 2 by adjusting each aeration amount of the mixed gas of air, oxygen, and nitrogen, and continues the culture.
- the cell concentration and the medium component concentration are measured by aseptically removing a part of the culture solution 7 during culture using the culture solution discharge pipe 11 or a separately provided sampling tube (not shown). Can do.
- the cell concentration is measured by optical turbidity, it is possible to connect a turbidity sensor provided in advance as the measurement means 15 described above. Thereby, during culture, the culture environment in the culture solution 7 can be maintained in a state appropriate for cell culture.
- the agitation drive device 5 is stopped and the swinging of the housing part 2 is stopped. Thereafter, the culture solution 7 is discharged through the culture solution discharge pipe 11 provided in the lower part of the culture bag 1. From the discharged culture solution 7, a series of culture production steps are completed by performing post-processes such as collection and purification of the target culture product. After the culture solution 7 is discharged, the culture bag 1 is removed from the housing portion 2 and discarded. In order to start the next culture production process, a new culture bag 1 is attached to the housing part 2.
- FIG. 8 is a schematic configuration diagram illustrating another embodiment of the cell culture device according to the present invention.
- the cell culture device 200 shown in FIG. 8 is different from the above-described cell culture device 100 in that the cell culture device 200 is provided with a curved convex protrusion 26 at the bottom of the housing portion 2.
- the configuration is exactly the same as that of the cell culture device 100. Therefore, regarding the cell culture device 200, the configuration different from the cell culture device 100 will be described, and the description of the same configuration as the cell culture device 100 will be omitted.
- FIG. 9A is a schematic diagram illustrating an example of a protrusion provided on the bottom of the housing. As shown in FIG. 9A, four protrusions 26 can be arranged radially at equal intervals from the center position of the bottom of the housing part 2.
- FIG. 9B is a schematic diagram illustrating another example of the protrusion provided on the bottom of the housing. As shown in FIG. 9B, three protrusions 26 can be arranged radially at equal intervals from the center position of the bottom of the housing part 2. If the projections 26 are provided in the mode shown in FIG. 9A or the mode shown in FIG. 9B, in any case, the culture solution 7 being stirred can hit the projections 26 and further promote the flow in the vertical direction. Therefore, the cell culture device 200 can further improve the stirring efficiency of the culture solution 7.
- FIG. 10A is a perspective view for explaining a specific shape of the protrusion 26.
- FIGS. 10B to 10E are cross-sectional views illustrating specific shapes of the protrusions 26.
- the protrusion 26 can have a substantially triangular prism shape.
- 10A is different from the protrusion 26 shown in FIG. 10B in that the protrusion 26 shown in FIG. 10B has a larger radius of curvature of the side portion 26a than that shown in FIG. 10A.
- the protrusion part 26 can be made into a substantially semi-cylindrical shape as shown to FIG. 10C, and can be made into a substantially semi-elliptical cylinder shape as shown to FIG. 10D.
- the protrusion 26 may have an asymmetric shape in which the area of one surface 26c is different from the area of the other surface 26c.
- the stirring drive device 5 for the culture solution 7 is provided by a shaking method by rotating eccentrically from the central axis.
- the stirring method of the culture solution in the present invention is not limited to the above-described method, and various methods can be applied.
- FIG. 11 is a schematic configuration diagram illustrating another embodiment of the cell culture device according to the present invention.
- a cell culture device 300 shown in FIG. 11 shows an example in which a magnetic coupling is used for the stirring drive device 5.
- a top plate 51 is provided on the top of the culture bag 1 housed in the housing part 2.
- the magnetic coupling member 52 is arrange
- a rigid rotating shaft 24 is connected to the magnetic coupling member 52 and is immersed in the culture solution 7.
- a stirring blade 25 a and a stirring blade 25 b are fixed to the rotary shaft 24.
- the stirring drive device 5 when the stirring drive device 5 is driven in response to a signal from the control device 19, the magnetic coupling member 53 rotates. Then, the rotational force is transmitted to the rotary shaft 24 via the magnetic coupling member 52, and the stirring blade 25a and the stirring blade 25b rotate.
- the culture solution 7 can be stirred without providing a through hole in the culture bag 1.
- the sealing mechanism for stirring is not limited to the method using the magnetic coupling described above, and mechanical sealing means using a known mechanical seal (shaft seal device) can also be applied.
- the operation procedure of the culture in the cell culture apparatus 300 shown in FIG. 11 is the same as that of the cell culture apparatus 100 except that the flow stirring means of the culture solution 7 is different, and the description thereof will be omitted.
- the stirring efficiency of the culture solution can be improved.
- the projection does not require joining or holding tools and does not require an operation such as folding, the culture bag is not likely to be broken even if pressure is applied. Therefore, the concern that the culture solution leaks can be further reduced.
- the stirring efficiency of the culture solution is improved, the mixing of the cultured cells and the medium is promoted, the nutrient components for the cultured cells and the dissolved oxygen necessary for respiration are supplied and the culture production efficiency is improved. be able to.
- the stirring efficiency of the culture solution is improved, cell culture can be performed with less power than before, and the power cost can be reduced.
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Abstract
Description
しかしながら、特許文献1に記載されている発明のように、培養袋を構成するフィルムとともにバッフルをヒートシールして接合すると、バッフルに掛かる流体力などにより、接合部分に圧力がかかった際に当該接合部分が破れたり、剥がれたりして、培養液が漏出してしまうことが懸念される。また、接合温度や接合時間が適切に行われず、接合が不完全であったときも、当該接合が不完全な箇所から培養液が漏出してしまうことが懸念される。さらに、特許文献1に記載されている他の態様も、バッフル保持部によって保持されている部分に圧力がかかったときに培養袋が破れ、培養液が漏出してしまうことが懸念される。
なお、本発明に係る細胞培養装置および培養バッグは、医薬品や健康食品などの主原料となる物質を生産する微生物や動植物の細胞を培養する際に適用することができる。本発明において、生産対象の物質としては、何ら限定されるものではなく、例えば、抗体や酵素などのタンパク質、低分子化合物、高分子化合物などの生理活性物質、およびウイルスなどを挙げることができる。また、β-カロテンやアスタキサンチンなどのカロチノイド、クロロフィルやバクテリオクロロフィルなどの色素、食品または化粧品などの着色などに使用されるフィコシアニン等のフィコビリン蛋白質、脂肪酸などの生理活性物質を挙げることができる。
また、培養対象の細胞としては、動物細胞、植物細胞、光合成細菌、微細藻類、ラン藻類、昆虫細胞、細菌、酵母、真菌および藻類などを挙げることができる。特に、抗体や酵素などのタンパク質を生産する動物細胞を培養対象とすることが好ましい。また、培養に用いる培地についても特に限定されるものではなく、従来のあらゆる培地が使用可能である。
図1は、本発明に係る細胞培養装置の一実施形態を説明する構成概略図である。
図1に示すように、細胞培養装置100は、培養バッグ1を収めるハウジング部2を備えている。そして、このハウジング部2は、培養バッグ1と当接する当接面の一部に、湾曲凸状の突起部4、4が設けられている。なお、図1は、ハウジング部2内に培養液7が封入された培養バッグ1を収めた様子を図示している。また、図1に示すように、細胞培養装置100は、培養バッグ1に収められた培養液7を攪拌させる攪拌手段を備えている。攪拌手段として具体的には、ハウジング部2の下方に、支持架台3に固定された攪拌駆動装置5が設けてられている。この攪拌駆動装置5は、ハウジング部2を中心軸から偏心させて回転させることによって培養バッグ1に収められた培養液7を攪拌するものである。
ハウジング部2および支持架台3はいずれも、培養液7を充填した培養バッグ1の重さや圧力を支持して形状を維持できるだけの強度を有していればよく、金属製、硬質プラスチック製など材質を問わない。
ここで、図2は、図1に示す突起部4の一態様を説明するハウジング部2の切欠き図である。また、図3は、図1のIII-III線断面図である。なお、図3では、説明および図示の都合上、ハウジング部2の内壁から浮かせて培養バッグ1を図示しているが、実際には、封入された培養液7の圧力によってハウジング部2の内壁面や突起部4に圧着している。また、図4A~Cはそれぞれ、突起部4の他の態様を説明する断面図である。
図7に示すように、他の態様として、ハウジング部2内に1つ以上の穴部21aを有している固定機構21を設ける。そして、突起部4には、固定機構21の穴部21aに固定するための固定部20を設ける。
固定機構21は、固定部20によって突起部4を固定することができればどのようなものでもよいが、例えば、パンチングメタルや金網で形成することができる。
固定部20は、辺となる部分4aの対面側、すなわち突起部4の底辺に設けるのが好ましい。このようにすると、固定部20が培養バッグ1に接触して培養バッグ1が破れてしまうという事態を回避することができる。固定部20は、例えばパンチングメタルの穴に合う形状の凸状体や金網の網目に係止するフックなどとするとよい。
固定機構21と固定部20を備えることによって、突起部4の取り付け位置及び取り付け角度を自在に変更することができ、且つ、突起部4を着脱自在に固定することができる。
本発明に係る培養バッグ1は、図1に示すように、細胞培養装置100のハウジング部2に収められ、可撓性を有し、内部に培養液7を封入可能なものであれば特に限定されることなく使用可能である。
培養バッグ1は、例えば、エチレン・ビニル・アセテートやエチル・ビニル・アルコールなどの多層フィルムで構成されたものを好適に用いることができる。このような培養バッグ1は、医薬品包装用途のシングルユースのバッグが各社から市販されており、任意に選択して使用することができる。なお、市販されている培養バッグは、一般的に、予めガンマ線、紫外線、エチレンオキシドガスなどによって滅菌されて無菌状態が維持され、気体や液体などの内容物がほぼ抜かれて、折り畳まれた状態で提供されている。
液中通気用ガス供給管8の培養バッグ1内部に設けた先端部には、気泡を発生させるための散気手段13が接続されている。なお、散気手段13は、融着などにより培養バッグ1内の側壁部に沿った底部に設けられている。このように散気手段13を設けると、散気手段13によって生じた気泡によって培養液7に上昇流が生じる。従って、培養液7の攪拌効率をさらに向上させることができる。
また、液中通気用ガス供給管8、気相用ガス供給管9、培地供給管10、培養液排出管11、およびガス排出管12にはそれぞれバルブ6が設けられており、通気、通液のON/OFFやそれらの流量を適切に制御できるようになっている。
また、培養状態の計測手段として、pH、温度、溶存酸素濃度、および溶存炭酸ガス濃度などを計測する装置と、それぞれのセンサとを具備しているが、図1中には簡略化して計測手段15として記載した。
以上に説明した細胞培養装置100と培養バッグ1の操作方法(培養の操作手順)について、図1を参照して以下に詳細に説明する。なお、以下の説明は操作の概要について説明するものであり、必ずしも下記の手順に拘束されるものではない。
前記したように、支持架台3には、培養バッグ1とハウジング部2とを一体として、中心軸から偏心させて回転させることによる振盪方式による培養液7の攪拌駆動装置5が設けられている。攪拌駆動装置5を駆動させることによって、培養バッグ1内の培養液7を流動させ、攪拌することができる。図3に示すように、攪拌駆動装置5によって、培養液7は概ね水平方向の旋回流を生じるが、外周部の培養液7の一部は、ハウジング部2の突起部4(図3では4個の突起部4を例示している)によって形成されたバッフルに衝突する。これにより、旋回流の方向が転向されて上下方向の流れが生じ、培養液7が均一になるように、効率的に混合することができる。
培養液7が排出された後、ハウジング部2から培養バッグ1が取り外されて廃棄される。次の培養生産工程を開始するには、新たな培養バッグ1がハウジング部2に取り付けられる。
図8は、本発明に係る細胞培養装置の他の実施形態を説明する構成概略図である。図8に示す細胞培養装置200と前記した細胞培養装置100とは、細胞培養装置200がハウジング部2の底部に湾曲凸状の突起部26を設けていた点が相違しており、それ以外の構成は細胞培養装置100と全く同様である。従って、細胞培養装置200については、細胞培養装置100と相違する構成について説明し、細胞培養装置100と同様の構成については説明を省略する。
また、図9Bは、ハウジング部の底部に設けた突起部の他の一例を示す概略図である。図9Bに示すように、突起部26は、ハウジング部2の底部の中心位置から放射線状に等間隔で3つ配置することができる。
図9Aに示す態様や図9Bに示す態様で突起部26を設けると、いずれの場合も、攪拌されている培養液7が当該突起部26にあたり、上下方向の流れをさらに促すことができる。そのため、細胞培養装置200は、培養液7の攪拌効率をさらに向上させることができる。
突起部26は、図10Aや図10Bに示すように、略三角柱状とすることができる。なお、図10Aに示す突起部26と図10Bに示す突起部26は、図10Bに示す突起部26の方が、図10Aに示すものよりも辺となる部分26aの曲率半径が大きい点で異なっている。
また、突起部26は、図10Cに示すように、略半円柱状とすることができ、図10Dに示すように、略半楕円柱状とすることができる。さらには、突起部26は、図10Eに示すように、一方の面26cの面積と他方の面26cの面積が異なるような非対称の形状とすることもできる。
細胞培養装置100および細胞培養装置200では、培養バッグ1とハウジング部2とを一体として、中心軸から偏心させて回転させることによる振盪方式による培養液7の攪拌駆動装置5が設けられている例について説明した。しかしながら、本発明における培養液の攪拌方法は前記したものに限定されず、種々の方式を適用することができる。
図11に示す細胞培養装置300は、攪拌駆動装置5にマグネットカップリングを用いた例を示している。細胞培養装置300は、ハウジング部2内に収められた培養バッグ1の上部に天板51が設けられている。そして、培養バッグ1の内部に磁性カップリング部材52を配置し、天板51上の磁性カップリング部材53との間でマグネットカップリングを構築している。磁性カップリング部材52には、剛性を有している回転シャフト24が接続されており、培養液7中に浸されている。回転シャフト24には、攪拌翼25aおよび攪拌翼25bが固定されている。
本発明によれば、培養液の攪拌効率が向上するので、培養細胞と培地の混合を促進し、培養細胞に対する栄養成分、および呼吸に必要な溶存酸素の供給を行うとともに培養生産効率を向上させることができる。
また、培養液の攪拌効率が向上するので、従来よりも少ない動力で細胞培養を行うことができ、動力コストを低減することができる。
1 培養バッグ
1a 凹部
2 ハウジング部
4、26 突起部
Claims (14)
- 可撓性を有し、内部に培養液を封入可能な培養バッグを収めるハウジング部を備え、
前記ハウジング部の内面には、前記培養バッグと当接する当接面の一部に、湾曲凸状の突起部が設けられていることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記ハウジング部に前記培養バッグを収めたことを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記突起部の高さWが、前記ハウジング部の内寸Dに対して、W/Dで0.05~0.15の範囲にあることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記突起部が、鉛直方向と平行な方向に、又は、鉛直方向に対して傾斜させて、前記ハウジング部の側壁部に設けられていることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記突起部が、前記ハウジング部の底部に設けられていることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記ハウジング部内に、1つ以上の穴部を有している固定機構が設けられており、
前記突起部には、前記固定機構の穴部に固定するための固定部が設けられている
ことを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記ハウジング部が、前記培養バッグに収められた培養液を攪拌させる攪拌手段を備えていることを特徴とするシングルユース細胞培養装置。 - 請求項7において、
前記攪拌手段が、前記ハウジング部を中心軸から偏心させて回転させるものであるか、または、前記培養バッグ内に配置された攪拌翼によって攪拌されるものであることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記突起部の形状が、略三角柱状または略半円柱状であることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記突起部の高さが、当該突起部の底辺の長さの0.5~5倍であることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記突起部の辺の曲率半径が、当該突起部の底辺の長さの0.05~0.5倍であることを特徴とするシングルユース細胞培養装置。 - 請求項1において、
前記突起部の長手方向の長さLが、前記培養バッグに封入される培養液の液面高さHに対して、L/Hで0.05~1の範囲にあることを特徴とするシングルユース細胞培養装置。 - シングルユース細胞培養装置のハウジング部に収められ、可撓性を有し、内部に培養液を封入可能であり、
前記ハウジング部の一部に設けられた湾曲凸状の突起部と対応する形状の凹部を設けたことを特徴とする培養バッグ。 - 請求項13において、
気泡を発生させる散気手段を側壁部に沿った底部に設けたことを特徴とする培養バッグ。
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- 2016-07-01 US US15/749,609 patent/US11015159B2/en active Active
- 2016-07-01 SG SG11201800936WA patent/SG11201800936WA/en unknown
- 2016-07-01 CN CN201680045254.2A patent/CN107922906A/zh active Pending
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Also Published As
Publication number | Publication date |
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US20180223233A1 (en) | 2018-08-09 |
US11015159B2 (en) | 2021-05-25 |
CN107922906A (zh) | 2018-04-17 |
JP2017035009A (ja) | 2017-02-16 |
JP6605251B2 (ja) | 2019-11-13 |
SG11201800936WA (en) | 2018-03-28 |
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