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WO2024056037A1 - Dispositif d'aspiration de liquide, bioréacteur et procédé d'aspiration de liquide - Google Patents

Dispositif d'aspiration de liquide, bioréacteur et procédé d'aspiration de liquide Download PDF

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Publication number
WO2024056037A1
WO2024056037A1 PCT/CN2023/118903 CN2023118903W WO2024056037A1 WO 2024056037 A1 WO2024056037 A1 WO 2024056037A1 CN 2023118903 W CN2023118903 W CN 2023118903W WO 2024056037 A1 WO2024056037 A1 WO 2024056037A1
Authority
WO
WIPO (PCT)
Prior art keywords
liquid
bioreactor
microcarriers
blade
tank
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/118903
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English (en)
Chinese (zh)
Inventor
鄢晓君
刘伟
隗功业
吴亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Cytoniche Biotech Co Ltd
Original Assignee
Beijing Cytoniche Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Cytoniche Biotech Co Ltd filed Critical Beijing Cytoniche Biotech Co Ltd
Priority to CN202380066026.3A priority Critical patent/CN119866368A/zh
Publication of WO2024056037A1 publication Critical patent/WO2024056037A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • 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

Definitions

  • the invention relates to a liquid pumping device, a bioreactor including the liquid pumping device, and a method for using the liquid pumping device to pump liquid, and belongs to the technical field of cell biological reaction equipment.
  • Bioreactor refers to an appropriate environment or engineering equipment that provides biochemical reactions. It usually refers to the use of enzymes or organisms (microorganisms) to make the device have the function of simulating organisms, and can perform biochemical reactions outside cells. In the simulated Both aerobic and anaerobic reactions can be carried out during the process.
  • bioreactors can be divided into natural bioreactors and engineered bioreactors. Bioreactors are usually cylindrical in shape, ranging in volume from a few liters to several cubic meters, and are often made of stainless steel. According to the way of adding raw materials, bioreactors can be divided into batch reactors (batch reactor), fed batch reaction systems (fed batch) and continuous flow reactors (continuous reactor), such as continuous stirred tank reactors , chemostat, etc.
  • Cells grown in bioreactors can be suspended in liquid media or grown attached to the surface of microcarriers.
  • the bioreactor When conducting high-density cell culture, the bioreactor only has a limited tank volume, and the culture medium it carries cannot meet the growth needs of the cells at all times. Therefore, it is necessary to frequently pump out the consumed culture medium and replenish fresh culture medium to meet the high-density culture. Nutrient requirements of densely cultured cells. Perfusion culture allows liquid to be continuously drained from the tank in real time while fresh medium is replenished into the tank at the same flow rate.
  • the tank of the bioreactor has a discharge port and a feed port in its structure.
  • Perfusion culture maintains a constant volume of culture medium in the tank through the balance of discharge and feed, achieving timely replenishment of fresh culture medium and avoiding the need for Occurrence of tank overflow. Since perfusion culture involves continuous low-speed liquid pumping and liquid feeding while the stirred bioreactor is running and stirring, the liquid pumping device cannot affect the operation of the stirring slurry on the one hand, and on the other hand, it is necessary to extract the culture medium consumed by nutrients. At the same time, cells or microcarriers should not be extracted.
  • a cup-type sleeve with a porous structure is placed on the stirring shaft.
  • the liquid only partially submerges part of the rotating filter and passes through the liquid outlet.
  • the tube extracts liquid, and the porous structure intercepts the microcarriers outside the cup to achieve separation, and as the stirring shaft rotates, the microcarriers can also be thrown away from the cup sleeve to a certain extent.
  • the disadvantage of this perfusion method is that it is prone to clogging.
  • a multi-layer funnel-type liquid suction pipe is placed above the top cover of the bioreactor, that is, outside the bioreactor tank, and the microcarriers and liquid are stratified and diverted through a multi-layer funnel.
  • the microcarrier density is higher in the funnel.
  • the narrow mouth falls downwards, while the lighter liquid is sucked upwards by the suction force of the liquid.
  • the disadvantage of this liquid extraction method is that the microcarriers will be repeatedly sucked out of the tank into the liquid extraction tube outside the tank and then fall back down. For continuous perfusion, the cells on the microcarriers will repeatedly leave the culture environment in the tank and then flow out of the tank. Falling back into the tank, this may have a greater impact on some more fragile cells.
  • the invention provides a liquid pumping device for a stirred bioreactor, a bioreactor including the liquid pumping device, and a method for pumping liquid using the liquid pumping device.
  • the liquid pumping device can be installed in a tank of the bioreactor. inside the body, thereby preventing cells on microtissues (e.g. microcarriers, cell-containing microcarriers, microcarrier aggregates, cell-containing microcarrier aggregates, cell aggregates such as embryoid bodies or organoids) from escaping from the bioreactor tank internal training environment.
  • microtissues e.g. microcarriers, cell-containing microcarriers, microcarrier aggregates, cell-containing microcarrier aggregates, cell aggregates such as embryoid bodies or organoids
  • the present invention provides a liquid pumping device.
  • the liquid pumping device includes a liquid pumping tube assembly.
  • the liquid pumping tube assembly is inserted into a tank of a bioreactor.
  • the top end of the liquid pumping tube assembly is fixed. on the top cover of the bioreactor.
  • microtissue refers to a tiny tissue unit, as well as a plurality of microtissue units, such as the same or different microtissue units, which are formed in any shape by aggregation, combination, or connection. Aggregates of unlimited density. Tiny tissue units include microcarriers, biological tissue units such as microorganisms such as bacteria, cells, etc.
  • Aggregations of multiple tiny tissue units include microcarrier aggregates; biological tissue aggregates, such as cell clumps formed by the aggregation of multiple identical or different cells, for example, the size of cell clumps is tens to hundreds of microns. , such as embryoid bodies, embryoid bodies of pluripotent stem cells, organoids, etc.; for example, multiple identical or different microorganisms such as bacteria aggregate to form bacterial masses; aggregates of biological tissues and microcarriers, such as microorganisms combined with cells Carriers, microcarrier aggregates bound to cells.
  • the bioreactor includes microtissues present in a liquid, such as microcarriers, cell-containing microcarriers, microcarrier aggregates, cell-containing microcarrier aggregates, cell clusters such as embryoid bodies. or organoids, bacterial clumps.
  • the liquid extraction tube assembly is detachably connected to the top cover of the bioreactor.
  • the liquid suction pipe assembly includes a liquid suction pipe, the liquid suction pipe includes a liquid outlet pipe body and a main body, and the liquid outlet pipe body is located above the main body.
  • the inner diameter of the liquid outlet pipe body is smaller than the inner diameter of the main pipe body.
  • the inner diameter of the liquid outlet pipe body is smaller than the inner diameter of the main pipe body, which is beneficial to smooth settlement.
  • the liquid extraction tube assembly is inserted into the tank of the bioreactor, and the top end of the liquid extraction tube assembly is fixed on the top cover of the bioreactor, that is, the liquid extraction tube assembly is completely Insert into the tank of the bioreactor.
  • the liquid suction pipe assembly is inserted into the tank of the bioreactor, for example, the liquid suction pipe assembly is partially inserted into the tank of the bioreactor.
  • the main pipe of the liquid suction pipe assembly may penetrate through the top cover of the bioreactor, so that the bottom opening of the main pipe is located in the tank, and the top and outlet of the main pipe The liquid pipe body is located outside the tank.
  • the liquid extraction tube assembly further includes a tapered opening located at the bottom of the main body.
  • the liquid suction pipe further includes a blade fixing rod, the blade fixing rod is detachably fixed in the liquid suction pipe, and a plurality of blades are connected to the blade fixing rod.
  • the blades advantageously assist in the settling of microtissues and the cells thereon.
  • the length of the blade fixing rod is less than or equal to the length of the main body.
  • the blade fixing rod is fixed inside the main body through a bottom end connection.
  • the blade fixing rod is fixed inside the main body in a bottom end collision manner.
  • angles between the plurality of blades and the blade fixing rod are equal.
  • an angle between the vertical direction of the plurality of blades and the blade fixing rod is an acute angle.
  • the plurality of blades are arranged perpendicularly to the blade fixing rod.
  • the plurality of blades are evenly distributed on the blade fixing rod.
  • the plurality of blades are divided into a plurality of blade groups, each blade group includes a plurality of blades, and the multiple blades of each blade group are located on the same side of the main rod through a blade root. on the horizontal plane and connected to the blade fixing rod.
  • the present invention also provides a bioreactor, which includes the liquid pumping device described in the first aspect and any embodiment thereof.
  • a bioreactor which includes the liquid pumping device described in the first aspect and any embodiment thereof.
  • the use of traditional micropore filtration methods is avoided, which can effectively avoid the occurrence of clogging, so that the culture medium can be continuously extracted for a long time. liquid; at the same time, since the liquid suction tube assembly is inserted into the tank of the bioreactor, the cells on the microtissue entering or leaving the liquid suction tube assembly will not cause the cells on the microtissue to leave the culture environment in the bioreactor tank, and then Avoid having a greater impact on fragile cells.
  • the bioreactor may be a bioreactor that utilizes microcarriers to culture cells, including microtissues such as microcarriers containing cells, present in a liquid.
  • the bioreactor includes microtissues such as microcarriers, cell-containing microcarriers, microcarrier aggregates, cell-containing microcarrier aggregates, cell clusters such as embryoid bodies or quasi-embryonic cells present in a liquid. organ.
  • Cell clusters are cell clusters with a size of tens to hundreds of microns, formed by the aggregation of multiple cells, such as embryoid bodies of pluripotent stem cells, whose size and shape are similar to microcarriers, cell-containing microcarriers, microcarrier aggregates, Microcarrier aggregates containing cells are similar.
  • the bioreactor further includes a stirring component and a liquid inlet pipe, and the liquid extraction pipe assembly, the stirring component and the liquid inlet pipe are arranged at intervals.
  • the liquid pumping process of the bioreactor will not affect the normal operation of the stirring assembly.
  • the horizontal position of the liquid inlet of the liquid inlet pipe is higher than the horizontal position of the bottom opening of the liquid extraction pipe.
  • the present invention also provides a liquid extraction method using the liquid extraction device described in the first aspect and any embodiment thereof.
  • the method includes: inserting the liquid extraction tube assembly into the bioreactor.
  • the top end of the liquid pumping tube assembly is fixed on the top cover of the bioreactor; liquid is pumped through the liquid pumping device, and during the liquid pumping process, the microorganisms entering the liquid pumping tube assembly are The tissue settles in the suction tube assembly and finally breaks away from the suction tube assembly to remain in the tank, and the culture medium liquid leaves the bioreactor from the top of the suction tube assembly.
  • the use of The traditional micropore filtration method can effectively avoid the occurrence of clogging, so that the culture medium liquid can be extracted for a long time; at the same time, because the liquid suction tube assembly is inserted into the tank of the bioreactor, cells on the microtissue can enter or leave the suction liquid.
  • the tube components will not cause the cells on the microtissue to leave the culture environment in the bioreactor tank, thereby avoiding a greater impact on the more fragile cells.
  • the microtissue enters the liquid suction tube through the tapered opening, settles in the main body of the liquid suction tube, and then leaves the liquid suction tube through the tapered opening.
  • the vanes block microtissue from moving up in the main body of the aspiration tube.
  • the blades advantageously assist in the settling of microtissues and the cells thereon.
  • the microtissue is selected from the group consisting of microcarriers, cell-containing microcarriers, microcarrier aggregates, cell-containing microcarrier aggregates, cell aggregates such as embryoid bodies or organoids, and bacterial aggregates.
  • the present invention also provides the application of the liquid pumping device described in the first aspect and any embodiment thereof or the bioreactor described in the second aspect and any embodiment thereof in liquid pumping.
  • liquid pumping device Compared with the existing technology, the liquid pumping device, bioreactor and liquid pumping method provided by this application have the following beneficial effects.
  • the liquid suction pipe assembly is detachably connected to the top cover of the bioreactor, which facilitates the removal of the liquid suction pipe assembly from the top cover when maintenance or replacement is required. Replace the top cover, or replace the entire bioreactor.
  • the liquid suction pipe assembly includes a liquid suction pipe.
  • the liquid suction pipe includes a liquid outlet pipe body and a main body.
  • the liquid outlet pipe body is located above the main pipe body.
  • the inner diameter of the liquid outlet pipe body is smaller than the main pipe body.
  • the inner diameter of the main body is described above; the inner diameter of the liquid outlet pipe is smaller than the inner diameter of the main body, which is beneficial to the smooth progress of settlement.
  • Figure 1 shows an exploded schematic diagram of a bioreactor according to an embodiment of the present invention.
  • Figure 2 shows a schematic three-dimensional structural diagram of a liquid extraction device according to an embodiment of the present invention.
  • Figure 3 shows a schematic three-dimensional structural view of a liquid extraction device according to another embodiment of the present invention, in which the liquid extraction tube has a tapered opening.
  • Figure 4 shows a schematic three-dimensional structural diagram of the enhanced flow guide assembly of the liquid pumping device according to an embodiment of the present invention, where A is an exploded schematic diagram; B shows a schematic diagram of the installation of the enhanced flow guide assembly and the liquid pumping pipe.
  • Figure 5 shows a schematic structural diagram of the liquid suction pipe reinforcement assembly according to an embodiment of the present invention, in which A is a longitudinal cross-sectional view of the liquid suction pipe reinforcement assembly; B is a top view of the liquid suction pipe reinforcement assembly; C is a diagram of the blade and the blade fixing rod. hang down
  • the included angle between the vertical directions is 30°; the vertical included angle between the blades and the blade fixed rod in D is 45°; the vertical included angle between the blades and the blade fixed rod in E is 60°.
  • Figure 6 shows a schematic structural diagram of a liquid pumping device reinforcement assembly according to another embodiment of the present invention, in which the blades are circular and are arranged perpendicularly to the blade fixing rod.
  • A is a side view and B is a top view.
  • Figure 7 shows a physical diagram of a different liquid extraction device according to an embodiment of the present invention, including a liquid extraction tube and a reinforcement assembly.
  • Figure 8 shows that there are a large number of microcarriers in the discharge liquid obtained by using the discharge pipeline of the prior art to extract the microcarrier suspension in the bioreactor.
  • Figure 9 shows that using different liquid extraction devices according to embodiments of the present invention to operate at a flow rate of 3.52 mL/min for 30-180 minutes, none of the discharged liquid obtained by extracting the microcarrier suspension in the bioreactor was observed under a microscope.
  • the microcarriers are pumped out, where the numbers of the pumping devices correspond to those in Figure 7.
  • Figure 10 shows the discharge liquid obtained by extracting the microcarrier suspension in the bioreactor using different liquid extraction devices according to the embodiment of the present invention operating at a flow rate of 3.52 mL/min for 19-48 hours.
  • the microcarriers are observed under a microscope.
  • the pumped-out case, in which microcarriers were observed at 19 and 48 hours using a short booster assembly, is indicated by the arrow in the figure, and the number of the pumping device corresponds to that in Figure 7.
  • Figure 11 shows the results obtained by using different liquid extraction devices according to embodiments of the present invention to extract the microcarrier suspension in the bioreactor by operating at flow rates of 3.52mL/min, 7.04mL/min and 10.56mL/min for 30-180 minutes. No microcarriers were observed to be pumped out in the discharged liquid under a microscope, and the number of the liquid pumping device corresponds to Figure 7.
  • Figure 12 shows the cell proliferation multiples and changes in glucose content in the culture system for stem cell three-dimensional porous microcarrier perfusion culture in a stirred bioreactor using a liquid extraction device according to an embodiment of the present invention.
  • Figure 13 shows that on the 3rd, 4th and 5th day of stem cell three-dimensional porous microcarrier perfusion culture in a stirred bioreactor using a liquid extraction device according to an embodiment of the present invention, there was no obvious abnormality in the extracted liquid. Microcarriers are expelled.
  • Figure 14 shows that the liquid in the outlet hose on day 5 of stem cell three-dimensional porous microcarrier perfusion culture in a stirred bioreactor using a liquid extraction device according to an embodiment of the present invention is still full, and no vacuoles are found.
  • Figure 15 shows the cell proliferation fold and changes in glucose content in the culture system of VERO cells perfused on three-dimensional porous microcarriers in a stirred bioreactor using a liquid extraction device according to an embodiment of the present invention.
  • Figure 16 shows that during the three-dimensional porous microcarrier perfusion culture of VERO cells in a stirred bioreactor using a liquid extraction device according to an embodiment of the present invention, the liquid discharged from the liquid outlet to the waste bottle is clear waste. liquid, no microcarriers are discharged.
  • Figure 17 shows a physical diagram of a different liquid extraction device according to an embodiment of the present invention, including a liquid extraction tube and a reinforcement assembly.
  • Figure 18 shows a schematic structural diagram of a liquid extraction device reinforcement assembly according to another embodiment of the present invention, in which the blades are elliptical and are arranged obliquely to the blade fixing rod.
  • Figure 19 shows a schematic structural diagram of an enhanced assembly of a liquid pumping device according to another embodiment of the present invention, in which the blades are in a spiral shape.
  • Figure 20 shows a schematic structural diagram of a liquid pumping device reinforcement assembly according to another embodiment of the present invention, in which the blades are elliptical and are arranged obliquely to the blade fixing rod.
  • Figure 21 shows a schematic structural diagram of an enhanced assembly of a liquid extraction device according to another embodiment of the present invention, in which the blades are in a spiral shape.
  • Figure 22 shows a schematic structural diagram of a liquid pumping device reinforcement assembly according to another embodiment of the present invention, in which the blades are elliptical and are arranged obliquely to the blade fixing rod.
  • Figure 23 shows a schematic structural diagram of an enhanced assembly of a liquid pumping device according to another embodiment of the present invention, in which the blades are in a spiral shape.
  • Figure 24 shows a schematic structural diagram of a liquid pumping device reinforcement assembly according to another embodiment of the present invention, in which the blades are elliptical and are arranged obliquely to the blade fixing rod.
  • Figure 25 shows a schematic structural diagram of an enhanced assembly of a liquid pumping device according to another embodiment of the present invention, in which the blades are in a spiral shape.
  • Figures 26 and 27 show the test results of different perfusion tubes under different conditions.
  • Figure 28 shows the morphology of iPSC perfusion culture in a 2L reactor.
  • Figure 29 shows the proliferation effect of iPSC perfusion culture in a 2L reactor.
  • microcarriers specifically, they can be microcarriers, cell-containing microcarriers, microcarrier aggregates and/or cell-containing microcarrier aggregates.
  • this embodiment provides a liquid extraction device that can realize microcarrier perfusion culture.
  • the liquid extraction device includes a liquid extraction tube assembly.
  • the top of the liquid extraction tube assembly is fixed on the biological reaction device through a mounting screw 5.
  • the liquid outlet of the liquid outlet body 7 of the liquid suction pipe 3 is outside the tank of the bioreactor.
  • the main body 6 and the bottom opening 8 of the liquid suction pipe assembly below the installation screw are inserted into the bioreactor.
  • the liquid suction pipe assembly includes a liquid suction pipe 3
  • the liquid suction pipe 3 includes a main body 6 and a liquid outlet pipe body 7 .
  • the main layered flow distribution body of the multi-layer funnel-type liquid extraction tube that is, the multi-layer funnel-type pipe body
  • the microcarriers will be repeatedly sucked out of the tank 1 into the main body of the pipette outside the tank and then back down.
  • the cells on the microcarriers are repeatedly separated from the culture environment in the tank and then returned to the tank. Falling back from the outside of the tank into tank 1 may have a greater impact on some fragile cells.
  • the liquid extraction tube assembly is inserted into the tank 1 of the bioreactor, so that the cells on the microcarriers entering or leaving the liquid extraction tube assembly will not cause the cells on the microcarriers to leave the biological reaction.
  • the culture environment in the vessel 1 is thereby prevented from having a greater impact on the fragile cells.
  • the liquid suction pipe assembly has a large inner diameter, and the liquid suction pipe assembly uses the exclusion method to separate solids and liquids regardless of the pore size, it can effectively avoid clogging.
  • the liquid outlet pipe body 7 of the liquid suction pipe assembly is fixed on the top cover 2 of the bioreactor, and the liquid outlet of the liquid outlet pipe body 7 of the liquid suction pipe assembly passing through the top cover 2 is connected through a tubular structure such as a silicone hose.
  • a tubular structure such as a silicone hose.
  • extraction power equipment such as peristaltic pumps Set.
  • the culture medium liquid at a lower position inside the bioreactor tank 1 can be extracted through the liquid extraction pipe assembly.
  • the extraction power device is preferably a peristaltic pump.
  • the peristaltic pump includes a pump tube.
  • the culture medium liquid can be isolated in the pump tube.
  • the pump tube is replaceable and the culture medium liquid can flow reversely.
  • the pump tube can also be run dry. The maintenance cost of the peristaltic pump is low.
  • the tubular structure connecting the liquid outlet of the liquid extraction tube assembly and the extraction power device is preferably a silicone hose or a thermoplastic tube suitable for the extraction power device.
  • the silicone hose has a wide range of continuous use temperatures, is soft, safe and will not Contamination of culture medium liquid; thermoplastic tubes can be welded with pipes on other containers using a sterile welding machine to achieve aseptic operation and are suitable for disposable reactors or disposable cell culture processes.
  • the stirring assembly can be used together with the liquid suction pipe assembly.
  • the liquid suction pipe assembly will not affect the work of the stirring assembly, thus helping to prevent the perfusion process of the bioreactor from affecting the normal operation of the stirring assembly. . Therefore, the bioreactor using the liquid pumping device of this embodiment may be a stirred bioreactor.
  • the microcarriers, the cells on them, and the culture medium liquid all enter the main body 6 of the pipette assembly.
  • the outer wall of the main body 6 blocks the liquid disturbance generated when the bioreactor is stirred, so the liquid entering the main body 6
  • the liquid is relatively stationary, and the fluid force generated is not enough for the microcarriers to resist the upward disturbance of gravity, so the microcarriers settle downward to achieve separation of solid and liquid.
  • the microcarriers and the cells on them settle in the pipette assembly and leave the pipette assembly from the bottom opening 8 of the pipette assembly, while the culture medium liquid leaves the organism from the outlet of the pipette assembly.
  • the tank 1 of the reactor enters the hose and the peristaltic pump, thereby realizing the extraction of the culture medium liquid, and achieving the separation of the culture medium and the microcarrier during the extraction process.
  • the size of the liquid pumping device is related to the size of the tank 1, so there is usually no specific limit on the size of the liquid pumping device.
  • the tank 1 The inner diameter is between 140-240mm
  • the height of the tank 1 is between 210-440mm
  • the position of the liquid suction pipe in the tank 1 is between 0.23-1.5L (those skilled in the art can understand that this means that the liquid suction pipe The height (the distance between the bottom of the liquid suction pipe and the bottom of the tank) corresponds to the height of the liquid level when the liquid volume in the tank 1 is 0.23-1.50L)
  • the length of the liquid suction pipe 3 in the tank 1 is 165- Between 410mm, such as between 195-305mm, such as between 195-406mm, such as between 195-410mm; such as 165mm, 170mm, 175mm, 180mm, 185mm, 190mm, 195mm, 200mm, 250mm, 300mm,
  • the length of the main body 6 is between 165-410mm, such as between 165-245mm, such as between 165-380mm, such as between 165-390mm; such as 165mm, 172mm, 200mm, 250mm, 255mm, 284mm, 300mm, 350mm, 372mm, 380mm, 390mm, 400mm, 410mm, and length values between any of the above values, or a range with any two of these values as endpoints, such as 165-245mm or 165-380mm.
  • the inner diameter of the main body 6 is between 15-40mm, such as between 22-24mm, between 22-39mm, such as 15mm, 16mm, 17mm, 18mm, 19mm, 20mm, 21mm, 22mm, 23mm, 24mm, 25mm, 26mm, 27mm, 28mm, 29mm, 30mm, 31mm, 32mm, 33mm, 34mm, 35mm, 36mm, 37mm, 38mm, 39mm, 40mm, and length values between any of the above values, or a range ending with any two of these values , such as 22-24mm or 22-39mm.
  • the length of the blade fixing rod is between 15-410mm, such as 24-240mm, such as 24-354mm, such as 15-390mm; such as 15mm, 24mm, 50mm, 100mm, 150mm, 200mm, 240mm, 250mm, 300mm, 350mm , 354mm, 380mm, 390mm, 400mm, 410mm, and length values between any of the above values, or by these values Any two of them are the range of endpoints, such as 24-240mm or 24-354mm.
  • the inner diameter of the liquid outlet pipe body 7 is between 3-8mm, such as 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, and the length value between any of the above values, or a range with any two of these values as endpoints, For example 4-6mm.
  • the size of the liquid extraction device is related to the size of the tank.
  • the above dimensional data provided by this disclosure are only exemplary and are not intended to limit the protection scope of the present invention.
  • the size of the liquid pumping device of the present invention can exceed the above-mentioned exemplary range, for example, the inner diameter of the main pipe 6 exceeds 40 mm, and for example, the inner diameter of the liquid outlet pipe body 7 exceeds 8 mm, and other components are similar.
  • the liquid pumping rate is related to the volume of tank 1, so as to achieve the purpose of achieving a total daily liquid change of 3-4 tank volumes.
  • the extraction of culture medium liquid and the separation process of culture medium and microcarriers avoid the use of traditional micropore filtration methods, which can effectively avoid the occurrence of clogging, so that the culture medium liquid can be continuously extracted for a long time. ;
  • the liquid suction tube assembly is inserted into the tank 1 of the bioreactor, the cells on the microcarriers entering or leaving the liquid suction pipe assembly will not cause the cells on the microcarriers to leave the culture environment in the bioreactor tank. This in turn avoids greater impact on more fragile cells.
  • the liquid extraction tube assembly is detachably connected to the top cover 2 of the bioreactor.
  • the liquid suction pipe assembly is detachably connected to the top cover 2 of the bioreactor, so that when the liquid suction pipe assembly needs maintenance or replacement, it is only necessary to remove the liquid suction pipe assembly from the top cover 2 without replacing the top cover 2. Or replace the entire bioreactor.
  • a thread is provided at the mounting thread 5 on the liquid outlet pipe body of the liquid suction pipe assembly so that it is threadedly connected to the threaded hole of the top cover 2 .
  • the liquid suction pipe assembly can also be passed through an inverted bolt, and the bolt is fixed on the liquid suction pipe assembly.
  • the threaded section of the bolt penetrates the top cover 2 from the bottom surface of the top cover 2 and passes through the top surface of the top cover 2 . Passing out, the liquid suction pipe assembly can be fixed on the top cover 2 by threading the nut with the threaded section of the bolt.
  • the liquid suction pipe assembly includes a liquid suction pipe 3.
  • the liquid suction pipe 3 includes a liquid outlet pipe body 7 and a main body 6.
  • the liquid outlet pipe body 7 is located above the main body 6.
  • the inner diameter of the liquid outlet of the liquid pipe body 7 is smaller than the inner diameter of the bottom opening 8 of the main pipe 6 .
  • the shape of the liquid outlet pipe body 7 is not particularly limited. It can be a straight pipe, or it can also form a curved portion according to the actual installation position.
  • the main body 6 is hollow cylindrical, and the main body part has a uniform inner diameter, and may be cylindrical, for example.
  • the bottom opening 8 is used to suck the culture medium mixed with microcarriers (and the cells loaded thereon) in the tank into the main body 6, and the main body 6 is used for the sedimentation of the microcarriers and the cells thereon.
  • the inner diameter of the main body 6 is large enough to ensure that there is a large enough space to realize the settlement of the microcarriers and the cells on them. At the same time, the space needs to be large enough to isolate the liquid flow caused by external stirring.
  • the inner diameter of the main body 6 is above 15 mm, such as above 16 mm, above 17 mm, above 18 mm, above 19 mm, above 20 mm, above 21 mm, or above 22 mm.
  • the liquid outlet of the liquid outlet pipe body 7 is connected to an extraction power device such as a peristaltic pump through a tubular structure such as a silicone hose. Therefore, the liquid outlet needs a smaller inner diameter to achieve flow restriction and to be connected to a tubular structure such as a silicone hose.
  • the flow rate of the culture medium in the outlet pipe body 7 is too high, the flow rate of the culture medium in the main body 6 will also be very high.
  • the settling time of the microcarriers in the main body 6 is too short and is not enough to complete the settlement and will be extracted.
  • the power unit is withdrawn. In practical applications, the specific flow rate varies according to the density of microstructures (such as microcarriers) and is adjusted accordingly.
  • the inner diameter of the liquid outlet pipe body 7 is equal to the inner diameter of the main pipe body 6, when the liquid is pumped through the liquid outlet of the liquid outlet pipe body 7, it will cause greater interference to the liquid in the main body 6, that is, it will cause a larger accident. Flow, the microcarriers cannot settle and will be extracted by the extraction power device.
  • the inner diameter of the liquid outlet pipe body 7 is smaller than the inner diameter of the main pipe body 6, which is beneficial to smooth settlement.
  • the ratio of the inner diameters of the liquid outlet pipe body 7 and the main pipe body 6 is 1:10 to less than 1:1, such as 1:10, 1:9, 1:8, 1:7, 1:6, 1: 5. 1:4, 1:3, 1:2, 1:1.5, and the length value between any of the above values, or the range with any two of these values as the endpoints, preferably 1:8 to 1:4 , 1:7 to 1:5, or 1:6.
  • the top of the main body 6 has a tapered portion, and the inner diameter gradually decreases to match the inner diameter of the liquid outlet pipe 7 for connection.
  • the top of the main body 6 and the liquid outlet pipe 7 are connected using a connecting component well known to those skilled in the art.
  • the connecting component has an opening on one side that is suitable for the inner diameter of the main body 6 and an opening on the other side.
  • the inner diameter of the liquid outlet 7 is suitable for the opening.
  • the bottom opening 8 of the liquid extraction tube 3 can also be connected to the tapered opening 9 located at the bottom of the main body 6 .
  • the tapered opening 9 prevents accumulation of microcarriers at the bottom of the main body 6 .
  • the size of the cross-section of the tapered opening 9 continuously decreases along the vertical direction.
  • the term "tapered” refers to a three-dimensional shaped structure having a top surface and non-parallel sidewalls that taper to a base surface with a small but non-zero area.
  • the tapered structures may have cross-sectional shapes of circles, triangles, squares, pentagons, hexagons, etc.
  • Exemplary pyramidal structures may be cones, pyramids, etc.
  • the liquid flow in the tank 1 can disperse the microcarriers at the bottom of the tapered opening 9 and prevent microcarriers from gathering.
  • the tapered opening 9 includes an outlet side connected to the main body 6 and a corresponding inlet side.
  • the culture medium suspension mixed with microcarriers enters and passes through the tapered opening 9 from the inlet side, and enters the main body 6 from the outlet side.
  • the inner diameter of the outlet side of the tapered opening 9 is consistent with the inner diameter of the main body 6 , and the inner diameter of the inlet side is smaller than the inner diameter of the main body 6 .
  • the ratio of the inner diameter of the inlet side of the tapered opening 9 to the inner diameter of the main body 6 is 1:10 to less than 1:1, such as 1:10, 1:9, 1:8, 1:7, 1:6 , 1:5, 1:4, 1:3, 1:2, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.19, 1:1.18, 1:1.17, 1:1.16, 1 :1.15, 1:1.14, 1:1.13, 1:1.12, 1:1.11, 1:1.10, 1:1.09, 1:1.08, 1:1.07, 1:1.06, 1:1.05, 1:1.04, 1:1.03 , 1:1.02, 1:1.01, and the length value between any of the above values, or the range with any two of these values as endpoints, preferably 1:2 to less than 1:1, 1:18 to 1:1.05 wait.
  • the tapered opening 9 since the size of the cross section of the tapered opening 9 is gradually reduced, after the reinforcement component is placed on the main body 6, the tapered opening 9 also plays a role in fixing the reinforcement component.
  • the liquid extraction tube 3 may also include an enhanced flow guide assembly (also referred to as an enhanced assembly in this article).
  • the reinforcement assembly includes a vertical blade fixing rod 10, a plurality of blades 11 fixed on the blade fixing rod, an optional assembly fixing rod 12 and a handle 13 at the bottom end of the blade fixing rod.
  • the blade fixing rod 10 is detachably fixed in the suction pipe 3, so that when the blade fixing rod 10 needs to be replaced or maintained, there is no need to replace the entire suction pipe 3.
  • the plurality of leaves 11 can block the microcarriers and the cells on them, which is beneficial to assist the settlement of the microcarriers and the cells on them.
  • the blades 11 facilitate the settling of the microcarriers and the cells thereon.
  • the length of the blade fixing rod 10 is less than or equal to the length of the main body 6 , and the blade fixing rod 10 is fixed inside the main body 6 through a bottom end connection.
  • the bottom end of the blade fixing rod 10 is connected to the bottom end of the main body 6 .
  • a suspension structure 14 is made on the bottom opening 8 of the liquid suction pipe 3.
  • the component fixing rod 12 can be hung on the suspension structure 14 to detachably fix the reinforcement assembly to the suction pipe. in liquid pipe 3.
  • the handle 13 of the reinforcement assembly is designed below the assembly fixing rod 12 so that the handle 13 is located outside the lower opening of the suction tube, making it easier to install and disassemble the reinforcement assembly by holding the handle 13.
  • Figure 4A is an explosion schematic diagram
  • Figure 4B shows a schematic diagram of the installation of the enhanced flow guide assembly and the liquid suction pipe.
  • one or more, such as 1, 2, 3, 4 or more assembly fixing rods 12 are provided on the bottom end of the blade fixing rod 10 or on the outer circumferential surface close to the bottom end.
  • the assembly fixing rod is a round rod.
  • the shape of the fixing rod of the assembly is not particularly limited and can be a shape known in the art, as long as it can play a fixing role.
  • the plurality of component fixing rods may be evenly distributed along the bottom end of the blade fixing rod 10 or on the outer circumferential surface near the bottom end.
  • the component fixing rod contacts the tapered opening 9 to fix the reinforcing component in the suction pipe 3 .
  • the handle 13 of the reinforcement assembly is designed below the assembly fixing rod 12 so that the handle 13 is located outside the lower opening of the suction tube, making it easier to install and disassemble the reinforcement assembly by holding the handle 13.
  • the length of the blade fixing rod 10 is such that the top end of the blade fixing rod contacts the top of the main body 6, and the bottom end of the blade fixing rod is connected with the bottom end of the main body or conflicts with the tapered opening 9, thereby fixing the reinforcement assembly in In the suction tube 3.
  • the length of the blade fixing rod 10 is less than or equal to the length of the main body 6, for example, 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 of the length of the main body 6 %, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less, and the length between any of the above values, or the range ending in any two of the above values.
  • the length of the blade fixing rod 10 may be 50% of the length of the main body 6 .
  • the length of the blade fixing rod 10 may be equal to the length of the main body 6 , or 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92% of the length of the main body 6 %, 91%, 90%, or a range with any two of the above values as endpoints, such as 90%-95%.
  • the blade 11 is perpendicular to the blade fixing rod 10 .
  • the vertical angle between the blade 11 and the blade fixing rod 10 is an acute angle.
  • the angle can be 5°, 10°, 15°, 20°, 25°, 30°, 35°. , 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, etc., and the length between any of the above values, or any two of the above values are
  • the range of the endpoint is preferably 30° to 60°, such as 30°, 45° or 60°.
  • 5A is a longitudinal cross-sectional view of the liquid suction pipe reinforcement assembly
  • 5B is a top view of the liquid suction pipe reinforcement assembly
  • the vertical angle between the blade and the blade fixed rod is 30°
  • 5D The vertical angle between the middle blade and the blade fixing rod is 45°
  • the vertical angle between the 5E middle blade and the blade fixing rod is 60°.
  • the blades can also play a blocking role and help sedimentation proceed.
  • the impact force of the collision between the blade 11 and the cells can be weakened, which is beneficial to protecting the cells.
  • the vertical angle (axis center line) between the blade 11 and the blade fixing rod 10 is 30°.
  • the vertical angle (axis center line) between the blade 11 and the blade fixing rod 10 is 45°.
  • the vertical angle (axis center line) between the blade 11 and the blade fixing rod 10 is 60°.
  • the vertical angle between the blade 11 and the blade fixing rod 10 can be changed according to specific needs.
  • the blades 11 are rectangular, circular, oval or spiral.
  • the vertical angle between the blade 11 and the blade fixing rod 10 can be changed according to specific needs.
  • the blade 11 may be arranged perpendicularly to the blade fixing rod 10 .
  • the blades 11 may be in the shape of a rectangle, a trapezoid, a polygon, a triangle, a circle, an ellipse, a cone, a fan shape, an umbrella shape, a curved surface, etc.
  • the blades 11 are in a spiral shape, that is, multiple blades jointly form a spiral blade with the blade fixing rod 10 as the axis; or in other words, the multiple blades are spirally distributed around the blade fixing rod 10 . Such a distribution method can more effectively exert the blocking effect of the blades 11 .
  • the size of the blade 11 is not particularly limited. It shall be based on the size of the main pipe.
  • the maximum width of the liquid suction pipe reinforcement assembly is smaller than the inner diameter of the main pipe, so that it can be arranged in the main pipe body. It is preferably the maximum width of the liquid suction pipe reinforcement assembly.
  • the width is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85% of the inner diameter of the main pipe , 84%, 83%, 82%, 81%, 80%, and the length between any of the above values, or the range with any two of the above values as endpoints, such as 90-99%, 95-99%.
  • the vertical angle between the blade 11 and the blade fixing rod 10 may be an acute angle, a right angle or an obtuse angle.
  • angles between the plurality of blades 11 and the blade fixing rod 10 may be equal or different.
  • the multiple blades 11 have the same shape or different shapes. Preferably, the multiple blades 11 have the same shape.
  • the plurality of blades 11 are evenly distributed on the blade fixing rod 10 . That is, the plurality of blades 11 are equally spaced in the vertical direction.
  • the plurality of blades 11 are non-uniformly distributed on the blade fixing rod 10 . That is, the spacing between the plurality of blades 11 in the vertical direction is not equal.
  • the density of the blades 11 on the blade fixing rod 10 gradually increases. That is, along the vertical direction, the distance between adjacent blades gradually decreases.
  • the blade 11 is connected and fixed to the blade fixing rod 10 through a fixing part, for example, through a fixing part installed on the edge of the blade.
  • the blade fixing rod 10 passes through the blade body of the blade 11 , for example, through the blade body of the blade 11 in a perforated manner, thereby fixing the blade 11 to the blade fixing rod 10 .
  • the blade fixing rod 10 can pass through any position of the blade 11 body, preferably through the geometric center of the shape of the blade 11 body, such as the center of a circular or elliptical blade, or the apex point of a cone or an umbrella.
  • the plurality of blades 11 are divided into multiple blade groups, each blade group includes a plurality of rectangular blades 11 , and the multiple rectangular blades 11 of each blade group are located on the blade fixing rod through the blade root (fixer). 10 and connected with the blade fixing rod 10.
  • each blade group may include the same number or a different number of blades 11 .
  • the shape of the blades 11 in the blade group may be a rectangle, a trapezoid, a polygon, a triangle, a circle, an ellipse, a fan, etc., or may be a cone, an umbrella, a curved surface, etc.
  • the blade set may include 2, 3, 4 or more blades 11.
  • each blade 11 in each set of blades 11 is evenly distributed around the blade fixing rod 10 .
  • the angle between the blades 11 is 180°.
  • the angle between the blades 11 is 120°.
  • the spacing between multiple blade groups in the vertical direction is equal; or along the vertical direction, the spacing between adjacent blade groups gradually decreases.
  • each blade 11 of two adjacent blade groups is arranged staggered in the circumferential direction.
  • the circumferential positions of the respective blades 11 of two adjacent blade groups are kept consistent.
  • the shapes of the plurality of blades 11 are the same or different.
  • the shapes of the plurality of blades 11 are the same.
  • this embodiment also provides a bioreactor, which includes the above-mentioned liquid pumping device.
  • the bioreactor also includes a top cover 2, a tank 1, a stirring assembly and a liquid inlet pipe 18.
  • the stirring assembly includes: a stirring shaft 4, which is fixed on the top cover 2 through a bearing seat; and , the stirring paddle 17 is distributed on the stirring shaft 4, the liquid inlet pipe is fixed on the top cover 2, and the lower end is inserted into the tank body.
  • the stirring assembly can also be disposed at the bottom of the tank 1, in which case the liquid suction pipe assembly and the liquid inlet pipe 18 are arranged to avoid each other.
  • the liquid suction pipe assembly, the liquid inlet pipe 18 and the stirring assembly are all arranged at intervals, that is, the liquid suction pipe assembly, the liquid inlet pipe 18 and the stirring assembly are far away from each other, so the liquid pumping and liquid feeding processes of the bioreactor are mutually exclusive. Does not interfere with and will not affect the normal operation of the mixing components.
  • the stirring assembly is arranged at the center of the tank 1, and the liquid suction pipe assembly and the liquid inlet pipe 18 avoid the setting of the stirring assembly.
  • the stirring shaft 4 is fixed on the top cover 2 through the bearing seat, so the stirring shaft 4 can rotate to achieve stirring.
  • the stirring paddle 17 is fixed on the stirring shaft 4, which helps to further improve the stirring efficiency.
  • the horizontal position of the lower end of the liquid inlet pipe 18 inserted into the tank body is higher than the horizontal position of the bottom opening 8 of the liquid suction pipe 3, which is beneficial to the replacement of fresh culture medium and culture medium with consumed nutrients.
  • the fresh culture medium will enter the tank through the lower end of the liquid inlet pipe 18 and will be sucked away by the liquid suction pipe.
  • the stirring shaft By setting the lower end of the liquid inlet pipe 18 at a higher level and the culture medium entering the tank above the bottom opening 8 of the liquid suction pipe 3, there is enough opportunity to be stirred by the stirring shaft and mixed with the culture medium consumed by nutrients. , thereby achieving the purpose of continuous perfusion of fresh culture medium into the tank.
  • This embodiment also provides a liquid extraction method using the above-mentioned liquid extraction device.
  • the liquid extraction method includes the following steps: the top end of the liquid extraction tube assembly is fixed on the top cover of the bioreactor, and the liquid extraction tube assembly is inserted into the bioreactor.
  • the liquid In the tank 1 of the device; the liquid is pumped through the liquid pumping device.
  • the microcarriers entering the liquid pumping tube assembly settle in the liquid pumping tube assembly and finally break away from the liquid pumping tube assembly to remain in the liquid pumping tube assembly.
  • the culture medium liquid leaves the bioreactor from the top of the liquid suction tube assembly.
  • the extraction of the culture medium liquid and the separation process of the culture medium and the microcarrier avoid the use of traditional micropore filtration methods, which can effectively avoid the occurrence of clogging, so that the culture liquid can be extracted for a long time;
  • the liquid extraction tube assembly is inserted into the tank 1 of the bioreactor, the cells on the microcarriers entering or leaving the liquid extraction tube assembly will not cause the cells on the microcarriers to leave the culture environment in the bioreactor tank, and then the cells on the microcarriers will not leave the culture environment in the bioreactor tank. Avoid having a greater impact on fragile cells.
  • the liquid pumping rate can be adjusted according to the size of the tank and the size of the corresponding liquid pumping device.
  • the liquid pumping rate can meet the liquid replacement needs of the tank. For example, for a 5L tank, the liquid replacement volume every 24 hours reaches more than 4L, that is, generally speaking, more than 80% of the tank volume, such as 85% Above, above 90%, above 100%, above 150%, above 200%, above 250%, above 300%, above 400%, above 500%, from which the fluid pumping rate that meets the fluid replacement needs can be calculated.
  • the fluid replacement requirements for each volume of tank are known in the art and can be readily determined by those skilled in the art.
  • the liquid pumping rate can be greater than the calculated liquid pumping rate based on actual needs.
  • the liquid pumping rate V 0 is calculated based on the 24-hour liquid replacement volume reaching 100% of the tank volume.
  • the liquid pumping rate is The rate V can be as high as 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 10 times.
  • the liquid pumping rate may also be smaller than the liquid pumping rate calculated above according to actual needs.
  • the liquid withdrawal rate is as high as 4mL/min, as high as 5mL/min, as high as 6mL/min, as high as 7mL/min, as high as 8mL/min, as high as 9mL/min, as high as 10mL/min, Up to 11mL/min, up to 12mL/min, up to 13mL/min, up to 14mL/min, up to 15mL/min, up to 16mL/min, up to 17mL/min, up to 18mL/min, up to 19mL/min, up to 20mL/min, up to 21mL/min, up to 22mL/min, up to 23mL/min, up to 24mL/min, up to 25mL/min, up to 26mL/min, up to 27mL/min min, up to 28mL/min, up to 29mL/min, up to 30mL/min, up to 35mL/min, up to 40mL/min, up to 45mL/min, up
  • the microcarriers enter the pipette 3 through the tapered opening 9 , settle in the main body 6 of the pipette 3 , and then leave the pipette 3 through the tapered opening 9 .
  • the tapered opening 9 prevents accumulation of microcarriers at the bottom of the main body 6 .
  • the size of the cross-section of the tapered opening 9 continuously decreases along the vertical direction.
  • the liquid flow in the tank 1 can disperse the microcarriers at the bottom of the tapered opening 9 and prevent microcarriers from gathering.
  • the opening of the tapered opening 9 is small, it can prevent the main body 6 from entering too much culture medium suspension mixed with microcarriers at one time, further avoiding the possibility of the microcarriers and the cells on them being extracted.
  • the blades 11 prevent the microcarriers from moving upward in the main tube 6 of the liquid suction tube 3 .
  • the plurality of leaves 11 can block the microcarriers and the cells on them, which is beneficial to assist the settlement of the microcarriers and the cells on them.
  • the blade 11 is fixed on the blade fixing rod 10 .
  • the blades 11 facilitate the settling of the microcarriers and the cells thereon.
  • This embodiment specifically exemplifies 7 types of liquid pumping devices, and the physical styles are shown in Figure 7 .
  • Specific size parameters are as shown in the table below. These geometric dimensions are related to the size of the bioreactor tank and are not limited to specific parameters.
  • the inner diameter of the bottom opening in the table is equal to the inner diameter of the main pipe, the bottom end of the main pipe is not connected to the tapered opening; when the inner diameter of the bottom opening is smaller than the inner diameter of the main pipe, the bottom end of the main pipe is connected to a tapered opening. At this time, the inner diameter of the bottom opening is The inner diameter of the tapered opening.
  • This embodiment uses a bioreactor of the present invention to conduct a liquid extraction experiment of microcarrier suspension.
  • the liquid pumping devices used in this bioreactor are the 1#, 3# and 5# liquid pumping devices in Example 1, that is, 1# uses a liquid pumping device alone, 3# uses a long reinforcement component (250mm), and 5# #Use short reinforcement components (152mm) in combination.
  • the reinforcement components use rectangular blades.
  • the angles between the blades 11 and the blade fixing rod 10 are both 60°.
  • This embodiment mainly tests whether the extraction of individual liquids can be achieved under a low-concentration porous microcarrier system without the microcarriers being blocked or pumped out.
  • These geometric dimensions are related to the size of the bioreactor tank.
  • the dimensions designed in this example match the stirred bioreactor (model: FTVS05, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the example and are not specific. parameter restrictions.
  • the liquid pumping device of the present invention can effectively prevent the microcarriers from being pumped out under the condition of continuous stirring in the bioreactor, and only the liquid is pumped out.
  • the maximum liquid pumping rate reaches 2.8mL/min, that is, It can complete about 4L of liquid replacement within 24 hours a day, which basically meets the liquid replacement needs of a 5L bioreactor.
  • the results further show that the use of reinforced components can further prevent microcarriers from being extracted, and the closer the length of the reinforced component is to the length of the main body, the better the extraction rate.
  • the short reinforcement component can draw liquid faster without discharging liquid than the liquid extraction device without reinforcement component, which can be increased to 3.29mL/min, while the long reinforcement component can further increase the liquid withdrawal rate to 4.7mL/min.
  • the faster the pumping rate the faster the cells can drain the medium consumed by nutrients, and the faster the cells can be filled with fresh medium, which is beneficial to the growth of the cells.
  • the experimental results also showed that no clogging occurred during the entire liquid pumping process, regardless of whether reinforced components were used.
  • This embodiment uses a bioreactor of the present invention to conduct a liquid extraction experiment of microcarrier suspension.
  • the liquid extraction device used in the bioreactor is the 2# to 7# devices in Example 1.
  • the main test is in the update Whether the extraction of individual liquids can be achieved in a high-concentration solid sphere microcarrier system without the microcarriers clogging or being pumped out.
  • These geometric dimensions are related to the size of the bioreactor tank.
  • the dimensions designed in this example match the stirred bioreactor (model: FTVS05, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the example and are not specific. parameter restrictions.
  • the peristaltic pump speed is set to 8rpm, and the corresponding pumping rate is 3.52mL/min, which is about 5L/day.
  • Continuously pump liquid, and at the same time replenish phosphate buffer solution into the bioreactor tank at the same liquid feeding rate to maintain the concentration of microcarriers in the tube body.
  • Figure 8 shows that there are a large number of microcarriers in the liquid extracted by the ordinary discharge pipeline.
  • Figures 9 and 10 show that devices 5# and 6# have trace amounts of microcarriers pumped out at 48 hours, device 7# has trace amounts of microcarriers pumped out at 19 hours, and the remaining devices have no carriers pumped out.
  • the results show that whether it is a wide mouth or a tapered mouth (2# vs. 3#), a rectangular blade or a round blade (2# vs. 4#), it can prevent the microcarriers from being pumped out.
  • longer reinforcement components (3# vs. 5#) are more effective than short reinforcement components, while the angle of the rectangular blade has no obvious effect on the effect (5# vs. 6#).
  • FIG. 11 shows that no microcarriers were pumped out at the pumping rates of 3.52mL/min, 7.04mL/min and 10.56mL/min for the 2# and 3# devices, while the 4# device did not pump out at the pumping rate of 3.52mL/min. No microcarriers were pumped out, but a large number of microcarriers were pumped out at the pumping rates of 7.04mL/min and 10.56mL/min, so the 2# and 3# devices are more suitable for high-rate pumping.
  • This embodiment uses a bioreactor of the present invention to perform perfusion culture of stem cells on three-dimensional porous microcarriers.
  • the bioreactor uses the 3# liquid pumping device in Example 1, that is, a liquid pumping device containing an enhanced component.
  • the blades of the reinforced component are rectangular blades with an angle of 60°, the length of the reinforced component is 250mm, and the length of the main body is 255mm.
  • the specific operations are as follows.
  • Example 1 Install the 3# liquid extraction device in Example 1 into the tank of the stirred bioreactor.
  • the tank is a bioreactor tank with a maximum working volume of 5L.
  • the height of the glass tank is 342mm and the diameter is 170mm.
  • a thermoplastic pipe with an inner diameter of 3.1 mm is installed at the liquid outlet of the liquid suction pipe 3 and the liquid inlet pipe 18 respectively, and the other ends of the two thermoplastic pipes are sealed with a welding sealing machine.
  • the tank is sterilized according to the conventional bioreactor tank method, that is, high-pressure sterilization at 121°C for 60 minutes.
  • thermoplastic tube of the sterile liquid storage bag and the thermoplastic tube of the liquid inlet pipe 18 of the bioreactor use a peristaltic pump to pump the culture medium suspension of the microcarriers and cells into the bioreactor tank. in the body.
  • thermoplastic tube of a new empty sterile liquid storage bag (used as a container for waste culture fluid, henceforth referred to as the waste liquid bag) with the thermoplastic tube on the liquid outlet of the liquid extraction device.
  • Bacteria welding start the perfusion program, that is, start the peristaltic pump connected to the outlet of the liquid extraction tube to pump liquid at 2.82mL/min.
  • start the peristaltic pump at the liquid inlet to replenish rich material from the feeding bag into the tank at 2.82mL/min. Nutrient-containing culture medium.
  • Figure 12 shows the changes in glucose content and cell number. Generally, as the number of cells increases, nutrients in the culture medium are consumed, represented by glucose, and their content decreases. However, the results showed that under the perfusion rate of 6 rpm, during the first 4 days of culture, as the number of cells increased, the glucose content did not decrease significantly (only from 33mM to 31.4mM). It can be seen that nutrients are removed by this liquid extraction device. The consumed culture medium is pumped out and replenished with fresh culture medium through the liquid inlet pipe to maintain sufficient nutrients in the culture system. From the 4th to the 5th day, the glucose content dropped significantly to 23.7mM, and at the same time, the cells experienced explosive growth.
  • Figure 13 shows the liquid discharged from the outlet of the liquid extraction device in the waste bag. During the three days of perfusion, no microcarriers were observed under the microscope at any of the three observation time points. Moreover, on the fifth day of culture (the third day of perfusion), Figure 14 shows that the pipeline is still filled with liquid, and no cavitation or foam appears, indicating that the liquid extraction device is not blocked.
  • This embodiment uses a bioreactor of the present invention to perform perfusion culture of VERO cells on three-dimensional porous microcarriers.
  • the bioreactor uses the 8# liquid extraction device in Example 1, that is, the liquid extraction device containing an enhanced component.
  • the blades of the reinforced component are rectangular blades with an angle of 60°, the length of the reinforced component is 162mm, and the length of the main body is 165mm.
  • the specific operations are as follows.
  • Example 1 Install the 8# liquid pumping device in Example 1 into the bioreactor tank 1 of the present invention.
  • the tank 1 is specifically used in this embodiment, it is designed as a bioreactor tank with a maximum working volume of 2L.
  • the height of the glass tank is 219 mm and the diameter is 140 mm.
  • thermoplastic pipe with an inner diameter of 3.1mm on the liquid outlet of the liquid pipe 3 and the liquid inlet pipe 18 of the liquid pumping device, and seal the other ends of the two thermoplastic pipes with a welding sealing machine.
  • the microcarriers settle naturally.
  • a peristaltic pump was used to discharge the PBS supernatant from the glass jar and pump 1L culture medium into it. After stirring evenly, stop stirring again to allow the microcarriers to settle naturally, and pump out the basal medium supernatant again. At this time, the volume of the microcarriers and M199 suspension remains about 300mL.
  • thermoplastic tube of the sterile feeding bottle and the thermoplastic tube of the liquid inlet pipe 18 of the bioreactor use a peristaltic pump to pump the cell and culture medium suspension into the bioreactor tank.
  • start the perfusion program that is, start the peristaltic pump connected to the outlet of the liquid extraction tube, pump the liquid into the waste bottle at 6 rpm (i.e. 2.82mL/min), and start the peristaltic pump at the liquid inlet at the same time.
  • This speed can achieve 4L of medium replacement a day, that is, 2 tank volumes of culture medium can be replaced a day.
  • microcarriers containing cells from the bioreactor as samples every day for cell number detection and glucose content detection, where changes in glucose content represent changes in nutrient content.
  • Figure 15 shows the changes in glucose content and cell number. From day 0 to day 3, in the absence of perfusion, as the number of cells increases, nutrients in the culture medium are consumed, represented by glucose, and their content decreases. After the perfusion started on the 3rd day, although the cells had explosive growth, the glucose did not drop off a cliff, but remained at a certain concentration (above 16mm). It can be seen that the culture medium consumed by nutrients through this liquid extraction device Extracting and replenishing fresh culture medium through the liquid inlet tube can maintain sufficient nutrients in the culture system. The continuous increase in the number of cells also proves that the microcarriers containing cells have not been discharged from the tank. Otherwise, if they are pumped out of the tank with perfusion, the number of cells will continue to decrease.
  • Figure 16 shows that the liquid discharged from the liquid outlet of the liquid extraction device is clear waste liquid, and no microcarriers are discharged.
  • the inner diameter of the bottom opening in the table is smaller than the inner diameter of the main pipe, it is because the bottom end of the main pipe is connected to a tapered opening. At this time, the inner diameter of the bottom opening refers to the inner diameter of the tapered opening.
  • This embodiment uses a bioreactor of the present invention to conduct a liquid extraction experiment of microcarrier suspension.
  • the liquid pumping devices used in this bioreactor are the 9# and 10# liquid pumping devices in Example 6. That is, the 9# liquid pumping device uses an elliptical reinforced component (156mm, as shown in Figure 18), and the 10# uses a spiral blade. Reinforcement component (156mm, as shown in Figure 19).
  • This example mainly tests which one has a better perfusion effect when cultured in a 2L reactor microcarrier system: an elliptical reinforcement component or a spiral blade reinforcement component.
  • These geometric dimensions are related to the size of the bioreactor tank. The dimensions designed in this embodiment match the stirred bioreactor used in the embodiment and are not limited to specific parameters.
  • DMEM medium is a product of Shanghai Yuanpei Biotechnology Co., Ltd., product number: L120KJ, and is a conventional commercial product.
  • Fetal bovine serum is a product of Vicente Biotechnology Co., Ltd., product number: 086-150, and is a conventional commercial product.
  • the PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercial product.
  • Recombinant trypsin digestion solution is a product of Lanzhou Minhai Company, product number: S342KJ, and is a conventional commercial product.
  • Vero cells are African green monkey kidney cells, which are common cells in this field.
  • Microcarrier preparation Weigh 4.5g of microcarriers, add 1000ml of PBS solution, and sterilize by moist heat at 121°C for 30 minutes. After the microcarriers are cooled, add 1.0 LDMEM culture medium. After stirring thoroughly, sediment the microcarriers and discard the supernatant.
  • Cell seeding Use recombinant protease digestion solution to digest freshly cultured vero cells, resuspend the cells in DMEM medium containing 10% newborn calf serum, seed the cells at a density of 300,000-500,000/ml, and adjust the culture volume to 1.5L. Bioreactor parameters and start culturing cells.
  • the elliptical reinforced component of the liquid extraction device of the present invention can effectively block the microcarriers from being extracted when the bioreactor is continuously stirred, and only liquid is extracted, with a maximum liquid extraction rate of 7.05mL/ min, there is no clogging or microcarrier extraction, and about 10L of liquid replacement can be completed within 24 hours a day, fully meeting the liquid replacement needs of a 2L bioreactor.
  • the extraction rate of the spiral enhancement component is as high as 7.05mL/min at the beginning, but as the culture proceeds, the extraction rate decreases to 2.35mL/min, indicating that the spiral enhancement component will accumulate microcarriers; the faster The higher the pumping rate, the faster the cells can discharge the nutrient-consuming medium, and the faster they can fill in fresh medium, which is beneficial to the growth of the cells.
  • This embodiment uses a bioreactor of the present invention to conduct a liquid extraction experiment of microcarrier suspension.
  • the liquid pumping device used in this bioreactor is the 11# liquid pumping device in Example 6, that is, the 11# liquid pumping device uses an elliptical reinforced component (266mm, as shown in Figure 20).
  • This example mainly tests the perfusion effect of the elliptical enhanced component under the culture of 5L reactor microcarrier system. These geometric dimensions are related to the size of the bioreactor tank. The dimensions designed in this example match the stirred bioreactor (model: FTVS05, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the example and are not specific. parameter restrictions.
  • microcarriers such as Cytodex or porous microcarriers.
  • DMEM medium is a product of Shanghai Yuanpei Biotechnology Co., Ltd., product number: L120KJ, and is a conventional commercial product.
  • Fetal bovine serum is a product of Vicente Biotechnology Co., Ltd., product number: 086-150, and is a conventional commercial product.
  • the PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercial product.
  • Recombinant trypsin digestion solution is a product of Lanzhou Minhai Company, product number: S342KJ, and is a conventional commercial product.
  • Vero cells are African green monkey kidney cells, which are common cells in this field.
  • Microcarrier preparation Weigh 9g of microcarriers, add 2000ml of PBS solution, and sterilize by moist heat at 121°C for 30 minutes. After the microcarriers are cooled, add 2.0 LDMEM culture medium. After stirring thoroughly, sediment the microcarriers and discard the supernatant.
  • Cell inoculation Use recombinant protease digestion solution to digest freshly cultured vero cells, resuspend the cells in DMEM medium containing 10% newborn calf serum, inoculate the cells at a density of 300,000-500,000/ml, and the culture volume is 3L. Adjust the biological Reactor parameters and start culturing cells.
  • the elliptical reinforced component of the liquid pumping device of the present invention can effectively block the microcarriers from being pumped out under the condition of continuous stirring of the bioreactor, and only the liquid is pumped out.
  • the maximum liquid pumping rate reaches 7.99mL/min, without clogging or clogging.
  • microcarriers are extracted, about 11.5L of liquid can be replaced within 24 hours a day, which basically meets the liquid replacement needs of a 5L bioreactor.
  • This embodiment uses a bioreactor of the present invention to conduct a liquid extraction experiment of microcarrier suspension.
  • the liquid pumping device used in this bioreactor is the 15# liquid pumping device in Example 6, that is, the 15# liquid pumping device uses an elliptical reinforced component (372mm, as shown in Figure 24).
  • This example mainly tests the perfusion effect of the elliptical enhanced component under the culture of 15L reactor microcarrier system. These geometric dimensions are related to the size of the bioreactor tank. The dimensions designed in this example match the stirred bioreactor (model: FTVS15, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the example and are not specific. parameter restrictions.
  • DMEM medium is a product of Shanghai Yuanpei Biotechnology Co., Ltd., product number: L120KJ, and is a conventional commercial product.
  • Fetal bovine serum is a product of Vicente Biotechnology Co., Ltd., product number: 086-150, and is a conventional commercial product.
  • the PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercial product.
  • Recombinant trypsin digestion solution is a product of Lanzhou Minhai Company, product number: S342KJ, and is a conventional commercial product.
  • Vero cells are African green monkey kidney cells, which are common cells in this field.
  • Microcarrier preparation Weigh 36g of microcarriers, add 4000ml of PBS solution, and sterilize by moist heat at 121°C for 30 minutes. After the microcarriers are cooled, add 8.0 LDMEM culture medium. After stirring thoroughly, sediment the microcarriers and discard the supernatant.
  • Cell inoculation Use recombinant protease digestion solution to digest freshly cultured vero cells, resuspend the cells in DMEM medium containing 10% newborn calf serum, inoculate the cells at a density of 300,000-500,000/ml, and the culture volume is 12L. Adjust the biological Reactor parameters and start culturing cells.
  • the elliptical reinforcement component of the liquid extraction device of the present invention can effectively prevent the microcarriers from being extracted when the bioreactor is continuously stirred, and only the liquid is extracted.
  • the maximum liquid extraction rate is 23mL/min, and there is no blockage or microcarrier extraction.
  • About 33L of liquid replacement can be completed within 24 hours a day, which basically meets the liquid replacement needs of a 15L bioreactor.
  • microcarriers used in the above embodiments can be microcarriers well known in the art, including commercially available products, such as solid ball microcarriers Cytodex (Cytiva), 3D microslides (Beijing Huakan Biotechnology Co., Ltd. company); or use microspheres with similar characteristics to microcarriers, such as glass microspheres, plastic microspheres, etc., as substitutes for microcarriers for testing.
  • This embodiment uses the bioreactor of the present invention to conduct iPSC embryoid body perfusion culture experiments.
  • the liquid pumping device used in this bioreactor is the 11# liquid pumping device in Example 6, that is, the 11# liquid pumping device uses an elliptical reinforced component (266mm, as shown in Figure 20).
  • This example mainly tests the perfusion effect of the elliptical enhanced component under the culture of iPSC embryoid body suspension system in a 2L reactor. These geometric dimensions are related to the size of the bioreactor tank. The dimensions designed in this example match the stirred bioreactor (model: FTVS02, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the example and are not specific. parameter restrictions.
  • mTeSR1 culture medium is a product of STEMCELL Technologies, product number: 85850, and is a conventional commercially available product.
  • ACCUTASE is a product of STEMCELL Technologies, product number: 07920, and is a conventional commercial product.
  • the PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercial product.
  • DMEM/F12 culture medium is a product of Gibco Company, product number: C11330500BT, and is a conventional commercial product.
  • Y27632 is a product of Sigma Company, product number: Y0503-5mg, and is a conventional commercially available product.
  • iPSC cells are induced pluripotent stem cells and are common cells in this field.
  • Cell seeding Use Accutase to digest freshly cultured iPSC suspension cells, resuspend the cells in mTeSR1 medium containing 10 ⁇ M Y27632, inoculate cells at a density of 5*10 5 /ml, and culture the volume to 800 mL. Adjust the reactor parameters and start at 60 rpm. Culture cells away from light.
  • the elliptical reinforced component of the liquid extraction device of the present invention can effectively prevent iPSC embryoid bodies from being extracted when the bioreactor is continuously stirred, and can realize perfusion culture of iPSC cells.
  • iPSCs are round and relatively uniform in shape and can proliferate more than three times in 4 days.
  • the liquid extraction device of the present invention is also suitable for cell clumps such as embryoid bodies.

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Abstract

La présente invention concerne un dispositif d'aspiration de liquide, comprenant un ensemble de tuyaux d'aspiration de liquide. L'extrémité supérieure de l'ensemble de tuyaux d'aspiration de liquide est fixée à un couvercle supérieur d'un bioréacteur, et l'ensemble de tuyaux d'aspiration de liquide est inséré dans le corps d'un réservoir du bioréacteur. Grâce au dispositif d'aspiration de liquide, pendant l'aspiration d'un milieu de culture liquide et la séparation d'un milieu de culture et d'un micro-tissu, les blocages peuvent être évités efficacement car un mode de filtrage traditionnel des micropores n'est pas utilisé, et le milieu de culture liquide peut donc être aspiré de manière continue pendant une longue période de temps. Parallèlement, comme l'ensemble de tuyaux d'aspiration de liquide est inséré dans le corps du réservoir du bioréacteur, les cellules du micro-tissu ne quitteront pas un environnement de culture dans le réservoir du bioréacteur lorsque les cellules du micro-tissu entrent ou sortent de l'ensemble de tuyaux d'aspiration de liquide, ce qui permet d'éviter une grande influence sur les cellules fragiles.
PCT/CN2023/118903 2022-09-14 2023-09-14 Dispositif d'aspiration de liquide, bioréacteur et procédé d'aspiration de liquide Ceased WO2024056037A1 (fr)

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JPH06209761A (ja) * 1993-01-19 1994-08-02 Toyobo Co Ltd 細胞培養装置
US20090280565A1 (en) * 2005-12-22 2009-11-12 Corporation De L'ecole Polytechique Montr'eal High-rate perfusion bioreactor
CN101085978A (zh) * 2007-06-27 2007-12-12 广州铭康生物工程有限公司 一种用于动物细胞灌流培养的过滤－沉降双结合灌流系统
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