Liquid extraction device, bioreactor and liquid extraction method

Abstract

The liquid pumping device comprises a liquid pumping pipe assembly, the top end of the liquid pumping pipe assembly is fixed on a top cover of a bioreactor, and the liquid pumping pipe assembly is inserted into a tank body of the bioreactor. By means of the liquid extraction device, in the extraction process of the culture medium liquid and the separation process of the culture medium and the micro-tissue, due to the fact that a traditional microfiltration mode is not used, blocking can be effectively avoided, and the culture medium liquid can be continuously extracted for a long time; meanwhile, the liquid extraction pipe assembly is inserted into the tank body of the bioreactor, so that the cells on the micro-tissues cannot leave the culture environment in the tank of the bioreactor when entering or leaving the liquid extraction pipe assembly, and the relatively fragile cells are prevented from being greatly influenced.

Description

Liquid extraction device, bioreactor and liquid extraction method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. CN202211117419.6 filed on September 14, 2022, the entire contents of which are incorporated herein by reference.

[0003] The invention relates to a liquid pumping device, a bioreactor comprising the liquid pumping device, and a method for pumping liquid using the liquid pumping device, belonging to the technical field of cell bioreactor equipment.

[0004] 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 carry out biochemical reactions outside the cell. In the simulation process, both aerobic reactions and anaerobic reactions can be carried out. In terms of source, bioreactors can be divided into natural bioreactors and engineered bioreactors. Bioreactors are usually cylindrical, with a volume ranging from a few liters to a few cubic meters, and are often made of stainless steel. According to the way the raw materials are added, bioreactors can be divided into batch reactors, fed batch reaction systems, and continuous flow reactors, such as continuous stirred tank reactors and chemostat reactors.

[0005] Cells grown in a bioreactor can be suspended in a liquid medium or attached to the surface of a microcarrier for growth. When cells are cultured at a high density, the bioreactor has only a limited tank volume, and the culture medium carried cannot always meet the growth needs of cells. Therefore, it is necessary to frequently extract the consumed culture fluid and replenish fresh culture fluid to meet the nutrient needs of high-density cultured cells. Perfusion culture can discharge the liquid from the tank continuously in real time, and at the same time replenish fresh culture medium into the tank at the same flow rate. The tank of the bioreactor has a discharge port and a feed port in structure. Perfusion culture is to maintain the constant volume of the culture medium in the tank by balancing the discharge and feed, so as to achieve timely replenishment of fresh culture medium and avoid overflow. Since perfusion culture is a continuous low-speed extraction and inlet of liquid under the stirring of the stirred bioreactor, the device for extracting liquid should not affect the operation of the stirring paddle on the one hand, and on the other hand, cells or microcarriers should not be extracted while extracting the culture medium consumed by nutrients.

[0006] There are two methods of extracting liquid used in microcarrier perfusion culture bioreactors in the prior art.

[0007] First, a cup-shaped sleeve with a porous structure is placed on the stirring shaft to use a rotary filter. There is a liquid outlet pipe in the cup-shaped sleeve. The liquid only partially submerges a part of the rotary filter. The liquid is extracted through the liquid outlet pipe. The porous structure intercepts the microcarriers outside the cup to achieve separation. As the stirring shaft rotates, the microcarriers can also be thrown away from the cup-shaped sleeve to a certain extent. The disadvantage of this perfusion method is that it is easy to get blocked.

[0008] Secondly, a multi-layer funnel-type liquid extraction tube is set above the top cover of the bioreactor, that is, outside the bioreactor tank, and the microcarriers and liquid are layered and diverted through the multi-layer funnel. The microcarriers with higher density fall downward at the narrow mouth of the funnel, while the liquid with lower density is sucked upward by the suction force of the liquid extraction. The disadvantage of this liquid extraction method is that the microcarriers will be repeatedly sucked out of the tank body into the liquid extraction tube outside the tank and then fall back. For continuous perfusion, the cells on the microcarriers are repeatedly separated from the culture environment in the tank and then fall back from outside the tank into the tank body, which may have a greater impact on some relatively fragile cells.

[0009] Summary of the invention

[0010] The present invention provides a pumping device for a stirred bioreactor, a bioreactor comprising the pumping device, and a method for pumping liquid using the pumping device. The pumping device can be arranged inside a tank of the bioreactor, thereby preventing cells on microtissues (such as microcarriers, microcarriers containing cells, microcarrier aggregates, microcarrier aggregates containing cells, cell clusters such as embryoid bodies or organoids) from escaping from the culture environment in the bioreactor tank.

[0011] In the first aspect, the present invention provides a liquid extraction device, which includes a liquid extraction tube assembly, the liquid extraction tube assembly is inserted into the tank body of the bioreactor, and the top of the liquid extraction tube assembly is optionally fixed on the top cover of the bioreactor. Using the liquid extraction device, in the process of extracting the culture medium liquid and separating the culture medium from the microtissue, since the use of the traditional microporous filtration method is avoided, the occurrence of blockage can be effectively avoided, so that the culture medium liquid can be continuously extracted for a long time; at the same time, since the liquid extraction tube assembly is inserted into the tank body of the bioreactor, the cells on the microtissue will not leave the culture environment in the bioreactor tank when the cells enter or leave the liquid extraction tube assembly, thereby avoiding a greater impact on the more fragile cells. When used in this article, the term "microtissue" refers to a tiny tissue unit, and a plurality of tiny tissue units, such as the same or different multiple tiny tissue units, are aggregated, combined, combined or connected to form an aggregate of unlimited shape and unlimited density. The tiny tissue unit includes a microcarrier, a biological tissue unit such as a microorganism such as bacteria, a cell, etc. Aggregates of multiple micro-tissue units include microcarrier aggregates; biological tissue aggregates, such as cell clumps formed by aggregation of multiple identical or different cells, such as cell clumps with a size of 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 microcarriers combined with cells, microcarrier aggregates combined with cells. In some embodiments, the bioreactor includes microtissues present in the liquid, such as microcarriers, microcarriers containing cells, microcarrier aggregates, microcarrier aggregates containing cells, cell clumps such as embryoid bodies or organoids, and bacterial masses. In one embodiment of the first aspect, the liquid extraction tube assembly is detachably connected to the top cover of the bioreactor. Through this embodiment, it is beneficial to remove the liquid extraction tube assembly from the top cover when the liquid extraction tube assembly needs to be maintained or replaced, without replacing the top cover or replacing the entire bioreactor.

[0012] In one embodiment of the first aspect, the liquid extraction tube assembly includes a liquid extraction tube, and the liquid extraction tube includes a liquid outlet tube body and a main tube body, and the liquid outlet tube body is located above the main tube body.

[0013] In one embodiment of the first aspect, the inner diameter of the liquid outlet tube body is smaller than the inner diameter of the main tube body. According to this embodiment, the inner diameter of the liquid outlet tube body is smaller than the inner diameter of the main tube body, which is conducive to the smooth sedimentation.

[0014] In one embodiment of the first aspect, the liquid extraction tube assembly is inserted into the tank body 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 inserted into the tank body of the bioreactor.

[0015] In one embodiment of the first aspect, the liquid extraction tube assembly is inserted into the tank body of the bioreactor, for example, the liquid extraction tube assembly is partially inserted into the tank body of the bioreactor. When the liquid extraction tube assembly is partially inserted into the tank body of the bioreactor, the main body of the liquid extraction tube assembly can pass through the top cover of the bioreactor, so that the bottom opening of the main body is located in the tank body, and the top of the main body and the liquid outlet tube body are located outside the tank body.

[0016] In one embodiment of the first aspect, the liquid extraction tube assembly further comprises a tapered opening, and the tapered opening is located at the bottom of the main tube. This embodiment is helpful to prevent the microtissue from being extracted outside the tank.

[0017] In one embodiment of the first aspect, the liquid extraction tube further comprises a blade fixing rod, the blade fixing rod is detachably fixed in the liquid extraction tube, and a plurality of blades are connected to the blade fixing rod. Through this embodiment, the blades are helpful to assist the sedimentation of the microtissue and the cells thereon.

[0018] In one implementation of the first aspect, the length of the blade fixing rod is less than or equal to the length of the main pipe.

[0019] In one embodiment of the first aspect, the blade fixing rod is fixed inside the main pipe by means of a bottom end connection.

[0020] In an implementation manner of the first aspect, the blade fixing rod is fixed inside the main pipe body by a bottom end abutment.

[0021] In one embodiment of the first aspect, angles between the plurality of blades and the blade fixing rod are equal.

[0022] In an embodiment of the first aspect, an angle between the plurality of blades and a vertical direction of the blade fixing rod is an acute angle.

[0023] In one embodiment of the first aspect, the plurality of blades are arranged perpendicular to the blade fixing rod.

[0024] In one embodiment of the first aspect, the plurality of blades are evenly distributed on the blade fixing rod.

[0025] In one embodiment of the first aspect, the multiple blades are divided into multiple blade groups, each blade group includes multiple blades, and the multiple blades of each blade group are located on the same horizontal plane of the main rod through the blade roots and are connected to the blade fixing rod.

[0026] In a second aspect, the present invention further provides a bioreactor, which includes the liquid extraction device described in the first aspect and any embodiment thereof. Using this bioreactor, in the process of extracting the culture medium liquid and separating the culture medium from the microtissue, since the use of the traditional microporous filtration method is avoided, the occurrence of blockage can be effectively avoided, so that the culture medium liquid can be extracted continuously for a long time; at the same time, since the liquid extraction tube assembly is inserted into the tank body of the bioreactor, the cells on the microtissue entering or leaving the liquid extraction tube assembly 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. In some embodiments, the bioreactor can be a bioreactor that cultures cells using microcarriers, including microtissues present in liquid, such as microcarriers containing cells. In some embodiments, the bioreactor includes microtissues present in liquid, such as microcarriers, microcarriers containing cells, microcarrier aggregates, microcarrier aggregates containing cells, and cell clusters such as embryoid bodies or organoids. A cell cluster is a cell cluster with a size of tens to hundreds of microns, formed by the aggregation of multiple cells, such as an embryoid body of pluripotent stem cells. Its size and shape are similar to microcarriers, microcarriers containing cells, microcarrier aggregates, and microcarrier aggregates containing cells.

[0027] In one embodiment of the second aspect, the bioreactor further comprises a stirring assembly and a liquid inlet pipe, and the liquid extraction pipe assembly, the stirring assembly and the liquid inlet pipe are arranged at intervals. Through this embodiment, the liquid extraction process of the bioreactor will not affect the normal operation of the stirring assembly.

[0028] In one embodiment of the second aspect, 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. This embodiment is conducive to the replacement of fresh culture medium and culture medium consumed by nutrients.

[0029] In a third aspect, the present invention further provides a method for extracting liquid using the liquid extraction device described in the first aspect and any of its embodiments, the method comprising: inserting the liquid extraction tube assembly into the tank body of the bioreactor, and optionally fixing the top end of the liquid extraction tube assembly on the top cover of the bioreactor; extracting liquid through the liquid extraction device, during the liquid extraction process, the microtissue entering the liquid extraction tube assembly settles in the liquid extraction tube assembly and eventually detaches from the liquid extraction tube assembly to remain in the tank body, and the culture medium liquid leaves the bioreactor from the top of the liquid extraction tube assembly. Using this liquid extraction method, in the process of extracting the culture medium liquid and separating the culture medium from the microtissue, since the use of the traditional microporous filtration method is avoided, the occurrence of blockage can be effectively avoided, so that the culture medium liquid can be extracted for a long time; at the same time, since the liquid extraction tube assembly is inserted into the tank body of the bioreactor, the cells on the microtissue entering or leaving the liquid extraction tube assembly 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.

[0030] In one embodiment of the third aspect, the microtissue enters the liquid extraction tube through the tapered opening, settles in the main body of the liquid extraction tube, and then leaves the liquid extraction tube through the tapered opening. This embodiment is conducive to preventing the microtissue from being extracted outside the tank.

[0031] In one embodiment of the third aspect, the blade blocks the microtissue from moving up in the main body of the liquid extraction tube. Through this embodiment, the blade is conducive to assisting the sedimentation of the microtissue and the cells thereon.

[0032] In the present invention, the microtissue is selected from microcarriers, microcarriers containing cells, microcarrier aggregates, microcarrier aggregates containing cells, cell agglomerates such as embryoid bodies or organoids, and bacterial agglomerates.

[0033] Therefore, the present invention also provides the use of the liquid extraction device described in the first aspect and any embodiment thereof or the bioreactor described in the second aspect and any embodiment thereof in liquid extraction.

[0034] Compared with the prior art, the liquid pumping device, bioreactor and liquid pumping method provided in the present application have the following beneficial effects.

[0035] 1. By using the pumping device, in the process of extracting the culture medium liquid and separating the culture medium from the microtissue, since the use of the traditional microporous filtration method is avoided, the occurrence of blockage can be effectively avoided, so that the culture medium liquid can be continuously extracted for a long time; at the same time, since the pumping tube assembly is inserted into the tank body of the bioreactor, the cells on the microtissue entering or leaving the pumping tube assembly 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.

[0036] 2. The liquid extraction tube assembly is detachably connected to the top cover of the bioreactor, which is beneficial for only removing the liquid extraction tube assembly from the top cover when the liquid extraction tube assembly needs to be maintained or replaced, without replacing the top cover or the entire bioreactor.

[0037] 3. The liquid extraction tube assembly includes a liquid extraction tube, which includes a liquid outlet tube body and a main tube body. The liquid outlet tube body is located above the main tube body, and the inner diameter of the liquid outlet tube body is smaller than the inner diameter of the main tube body; the inner diameter of the liquid outlet tube body is smaller than the inner diameter of the main tube body, which is conducive to the smooth sedimentation.

[0038] The above technical features can be combined in various suitable ways or replaced by equivalent technical features as long as the purpose of the present invention can be achieved.

[0039] The present invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which like elements are numbered in a similar manner, and in which:

[0040] FIG. 1 shows a schematic exploded view of a bioreactor according to an embodiment of the present invention.

[0041] FIG. 2 shows a schematic three-dimensional structure diagram of a liquid pumping device according to an embodiment of the present invention.

[0042] FIG3 shows a schematic three-dimensional structure diagram of a liquid pumping device according to another embodiment of the present invention, wherein the liquid pumping tube has a tapered opening.

[0043] 4 shows a schematic diagram of the three-dimensional structure of the enhanced flow guiding component of the liquid pumping device according to an embodiment of the present invention, wherein A is an exploded schematic diagram; and B shows a schematic diagram of the installation of the enhanced flow guiding component and the liquid pumping tube.

[0044] Figure 5 shows a schematic structural diagram of a liquid extraction tube reinforcement assembly according to one embodiment of the present invention, wherein A is a longitudinal cross-sectional view of the liquid extraction tube reinforcement assembly; B is a top view of the liquid extraction tube reinforcement assembly; the vertical angle between the blade and the blade fixing rod in C is 30°; the vertical angle between the blade and the blade fixing rod in D is 45°; and the vertical angle between the blade and the blade fixing rod in E is 60°.

[0045] 6 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are circular and are arranged perpendicular to the blade fixing rods, A is a side view, and B is a top view.

[0046] FIG. 7 shows a pictorial diagram of a different pumping device including a pumping tube and a reinforcing assembly according to an embodiment of the present invention.

[0047] FIG8 shows that a large number of microcarriers are present in the effluent obtained by extracting a microcarrier suspension from a bioreactor using a discharge pipeline in the prior art.

[0048] FIG9 shows that no microcarriers were observed to be pumped out in the effluent obtained by extracting the microcarrier suspension in the bioreactor at a flow rate of 3.52 mL / min for 30-180 minutes using different pumping devices according to embodiments of the present invention, wherein the numbers of the pumping devices correspond to those in FIG7 .

[0049] Figure 10 shows the microcarriers being pumped out of the effluent obtained by extracting the microcarrier suspension in the bioreactor at a flow rate of 3.52 mL / min for 19-48 hours using different pumping devices according to embodiments of the present invention, where microcarriers were observed at 19 hours and 48 hours using a short reinforcement component, as indicated by the arrows in the figure. The numbering of the pumping devices corresponds to Figure 7.

[0050] Figure 11 shows that when different pumping devices according to embodiments of the present invention were operated at flow rates of 3.52 mL / min, 7.04 mL / min, and 10.56 mL / min for 30-180 minutes, no microcarriers were observed to be pumped out in the effluent obtained by extracting the microcarrier suspension in the bioreactor under a microscope, and the numbers of the pumping devices correspond to those in Figure 7.

[0051] FIG. 12 shows the cell proliferation multiples and the changes in the glucose content in the culture system of three-dimensional porous microcarrier perfusion culture of stem cells in a stirred bioreactor using a pumping device according to an embodiment of the present invention.

[0052] 13 shows that no obvious microcarriers were discharged in the extracted liquid on the 3rd, 4th and 5th days of three-dimensional porous microcarrier perfusion culture of stem cells in a stirred bioreactor using a liquid extraction device according to an embodiment of the present invention.

[0053] FIG. 14 shows that the liquid in the outlet hose is still full of liquid without cavitation on the 5th day of three-dimensional porous microcarrier perfusion culture of stem cells in a stirred bioreactor using a liquid pumping device according to an embodiment of the present invention.

[0054] FIG. 15 shows the cell proliferation multiples and the changes in glucose content in the culture system of VERO cells perfused cultured on three-dimensional porous microcarriers in a stirred bioreactor using a pumping device according to an embodiment of the present invention.

[0055] 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 into the waste liquid bottle is clear waste liquid, and no microcarriers are discharged.

[0056] FIG. 17 shows a pictorial diagram of a different pumping device including a pumping tube and a reinforcing assembly according to an embodiment of the present invention.

[0057] FIG. 18 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are elliptical and are arranged obliquely with respect to the blade fixing rod.

[0058] FIG. 19 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are spiral-shaped.

[0059] FIG. 20 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are elliptical and are arranged obliquely with respect to the blade fixing rod.

[0060] FIG. 21 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are spiral-shaped.

[0061] FIG. 22 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are elliptical and are arranged obliquely with respect to the blade fixing rod.

[0062] FIG. 23 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are spiral-shaped.

[0063] 24 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are elliptical and are arranged obliquely with respect to the blade fixing rod.

[0064] FIG. 25 shows a schematic structural diagram of a pumping device reinforcement assembly according to another embodiment of the present invention, wherein the blades are spiral-shaped.

[0065] Figures 26 and 27 show the test results of different perfusion tubes under different conditions.

[0066] FIG. 28 shows the morphology of iPSCs cultured in a 2 L perfusion reactor.

[0067] FIG. 29 shows the proliferation effect of iPSCs in perfusion culture in a 2L reactor.

[0068] List of reference numerals: 1-tank body; 2-top cover; 3-liquid extraction tube; 4-stirring shaft; 5-installation screw; 6-main body; 7-liquid outlet body; 8-bottom opening; 9-conical opening; 10-blade fixing rod; 11-blade; 12-assembly fixing rod; 13-handle; 14-suspension structure; 15-rectangular blade; 16-circular blade; 17-stirring paddle; 18-liquid inlet pipe

[0069] The technical solution of the present invention is further described in detail below through embodiments and in conjunction with the accompanying drawings, but the present invention is not limited to the following embodiments.

[0070] The following description is made by taking microcarriers (specifically, microcarriers, microcarriers containing cells, microcarrier aggregates and / or microcarrier aggregates containing cells) as an example, but it should be noted that these descriptions are also applicable to other microtissues.

[0071] As shown in FIG1 , this embodiment provides a liquid extraction device capable of realizing microcarrier perfusion culture, the liquid extraction device comprises a liquid extraction tube assembly, the top end of the liquid extraction tube assembly is fixed on the top cover 2 of the bioreactor through a mounting screw 5, so that the liquid outlet of the liquid outlet tube body 7 of the liquid extraction tube 3 is outside the tank body of the bioreactor, and the main body 6 and the bottom opening 8 of the liquid extraction tube assembly below the mounting screw are inserted into the tank body 1 of the bioreactor. Among them, the liquid extraction tube assembly comprises a liquid extraction tube 3, and the liquid extraction tube 3 comprises a main body 6 and a liquid outlet tube body 7.

[0072] In the background art, the main stratified flow dividing body of the multi-layer funnel-type liquid extraction tube, i.e., the multi-layer funnel-type tube body, is arranged above the bioreactor top cover 2, i.e., outside the bioreactor tank. The disadvantage of this liquid extraction method is that the microcarriers will be repeatedly sucked out of the tank body 1 into the main body of the liquid extraction tube outside the tank and then fall back. For continuous perfusion, the cells on the microcarriers are repeatedly separated from the culture environment in the tank and then fall back from the outside of the tank into the tank body 1, which may have a greater impact on some relatively fragile cells.

[0073] In order to prevent cells from escaping from the culture environment, the present embodiment inserts the liquid extraction tube assembly into the tank body 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 culture environment in the bioreactor tank body 1, thereby avoiding a greater impact on the more fragile cells.

[0074] At the same time, since the liquid extraction tube assembly has a larger inner diameter and the liquid extraction tube assembly does not rely on the pore size to separate the solid and the liquid using the exclusion method, the occurrence of blockage can be effectively avoided.

[0075] The liquid outlet tube body 7 of the liquid extraction tube assembly is fixed on the top cover 2 of the bioreactor, and the liquid outlet of the liquid outlet tube body 7 of the liquid extraction tube assembly passing through the top cover 2 is connected to an extraction power device such as a peristaltic pump through a tubular structure such as a silicone hose. When the extraction power device is working, the culture medium liquid at a lower position inside the bioreactor tank 1 can be extracted through the liquid extraction tube assembly.

[0076] The extraction power device is preferably a peristaltic pump, which includes a pump tube. The culture medium liquid can be isolated in the pump tube. The pump tube is replaceable, the culture medium liquid can be reversed, and the pump tube can also run dry. The peristaltic pump has low maintenance costs and other characteristics, which make the peristaltic pump have a competitive advantage over other extraction power devices.

[0077] The tubular structure connecting the liquid outlet of the liquid extraction tube assembly and the extraction power device is preferably a hose suitable for the extraction power device, such as a silicone hose or a thermoplastic hose. The silicone hose has a wide continuous use temperature range, is soft and safe, and will not cause contamination to the culture medium liquid; the thermoplastic tube can be welded to the pipes on other containers by a sterile welding machine to achieve sterile operation, and is suitable for disposable reactors or disposable cell culture processes.

[0078] In addition, since the perfusion culture uses a liquid extraction tube assembly, the stirring assembly can be used simultaneously with the liquid extraction tube assembly, and the liquid extraction tube assembly will not affect the operation of the stirring assembly, thereby helping to prevent the perfusion process of the bioreactor from affecting the normal operation of the stirring assembly. Therefore, the bioreactor using the liquid extraction device of this embodiment can be a stirring bioreactor.

[0079] During the extraction process, the microcarriers and the cells thereon as well as the culture medium liquid all enter the main body 6 of the liquid extraction tube 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 is relatively static, and the fluid force generated is not enough to allow the microcarriers to resist the upward disturbance of gravity, so the microcarriers settle downward to achieve the separation of solids and liquids. Using the principle of self-weight sedimentation, the microcarriers and the cells thereon settle in the liquid extraction tube assembly and leave the liquid extraction tube assembly from the bottom opening 8 of the liquid extraction tube assembly, while the culture medium liquid leaves the tank 1 of the bioreactor from the liquid outlet of the liquid extraction tube assembly and enters the hose and peristaltic pump, thereby achieving the extraction of the culture medium liquid and achieving the separation of the culture medium and the microcarriers during the extraction process.

[0080] The size of the liquid extraction device is related to the size of the tank body 1, so there is usually no specific restriction on the size of the liquid extraction device. In a specific embodiment, when the volume of the tank body 1 is 2-20L, optionally, the inner diameter of the tank body 1 is between 140-240mm, the height of the tank body 1 is between 210-440mm, and the position of the liquid extraction tube in the tank body 1 is between 0.23-1.5L (those skilled in the art will understand that this means that the height of the liquid extraction tube (the distance from the bottom end of the liquid extraction tube to the bottom of the tank) corresponds to the height of the liquid level when the volume of the liquid in the tank body 1 is 0.23-1.50L), and the length of the liquid extraction tube 3 in the tank body 1 is between 140-240mm and 210-440mm. It is between 165-410mm, for example, between 195-305mm, for example, between 195-406mm, for example, between 195-410mm; for example, 165mm, 170mm, 175mm, 180mm, 185mm, 190mm, 195mm, 200mm, 250mm, 300mm, 305mm, 350mm, 400mm, 406mm, 410mm, and length values ​​between any of the above values, or a range with any two of these values ​​as endpoints, for example, 195-305mm or 195-406mm. The length of the main body 6 is between 165-410mm, for example, between 165-245mm, for example, between 165-380mm, for example, between 165-390mm; for example, 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, for example, 165-245mm or 165-380mm. The inner diameter of the main body 6 is between 15 and 40 mm, for example, between 22 and 24 mm, between 22 and 39 mm, for example, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 37 mm, 38 mm, 39 mm, 40 mm, and length values ​​between any of the above values, or a range with any two of these values ​​as endpoints, for example, 22 to 24 mm or 22 to 39 mm.The length of the blade fixing rod is between 15-410mm, such as between 24-240mm, such as between 24-354mm, such as 15-390mm; such as 15mm, 24mm, 50mm, 100mm, 150mm, 200mm, 240mm, 250mm, 300mm, 350mm, 354mm, 380mm, 390mm, 400mm, 410mm, and any length value between the above values, or a range with any two of these values ​​as endpoints, such as 24-240mm or 24-354mm. The inner diameter of the liquid outlet tube body 7 is between 3-8mm, such as 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, and any length value between the above values, or a range with any two of these values ​​as endpoints, such as 4-6mm. Those skilled in the art should understand that the size of the liquid pumping device is related to the size of the tank body. The above-mentioned dimensional data provided in the present disclosure is only exemplary and is not intended to limit the scope of protection of the present invention. As the size of the tank body increases, the size of the liquid pumping device of the present invention may exceed the above-mentioned exemplary range. For example, the inner diameter of the main body 6 exceeds 40 mm, and the inner diameter of the liquid outlet tube body 7 exceeds 8 mm. Other components are similar.

[0081] During the process of pumping liquid, the pumping rate is related to the volume of the tank 1, so as to achieve the purpose of a total volume of 3-4 tanks of liquid replacement per day.

[0082] By utilizing the pumping device, the extraction of the culture medium liquid and the separation process of the culture medium and the microcarrier can effectively avoid blockage due to avoiding the use of the traditional microporous filtration method, thereby being able to continuously extract the culture medium liquid for a long time; at the same time, since the pumping tube assembly is inserted into the tank body 1 of the bioreactor, the cells on the microcarrier entering or leaving the pumping tube assembly will not cause the cells on the microcarrier to leave the culture environment in the bioreactor tank, thereby avoiding a greater impact on the more fragile cells.

[0083] In some embodiments, the liquid extraction tube assembly is detachably connected to the top cover 2 of the bioreactor.

[0084] The liquid extraction tube assembly is detachably connected to the top cover 2 of the bioreactor, so that when the liquid extraction tube assembly needs to be maintained or replaced, it is only necessary to remove the liquid extraction tube assembly from the top cover 2 without replacing the top cover 2 or the entire bioreactor.

[0085] Optionally, a thread is provided at the mounting screw port 5 on the liquid outlet tube body of the liquid extraction tube assembly so as to be threadedly connected with the threaded hole of the top cover 2 .

[0086] Optionally, the liquid extraction tube assembly can also be passed through an inverted bolt, which is fixed to the liquid extraction tube assembly. The threaded section of the bolt passes through the top cover 2 from the bottom surface of the top cover 2 and exits from the top surface of the top cover 2. The liquid extraction tube assembly can be fixed to the top cover 2 by using a nut to threadably connect with the threaded section of the bolt.

[0087] This embodiment facilitates that when the liquid extraction tube assembly needs maintenance or replacement, the liquid extraction tube assembly only needs to be removed from the top cover 2, without replacing the top cover 2 or the entire bioreactor.

[0088] In some embodiments, as shown in FIG2 , the liquid extraction tube assembly includes a liquid extraction tube 3, and the liquid extraction tube 3 includes a liquid outlet tube body 7 and a main tube body 6, wherein the liquid outlet tube body 7 is located above the main tube body 6, and the inner diameter of the liquid outlet of the liquid outlet tube body 7 is smaller than the inner diameter of the bottom opening 8 of the main tube body 6. The shape of the liquid outlet tube body 7 is not particularly limited, and can be a straight tube, or a curved portion can be formed according to the actual installation position. The main tube body 6 is a hollow column, and the main body has a uniform inner diameter, for example, it can be cylindrical.

[0089] 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 enough space to achieve the sedimentation of the microcarriers and the cells thereon, and the space needs to be large enough to isolate the liquid flow caused by external stirring. Optionally, the inner diameter of the main body 6 is above 15mm, for example, above 16mm, above 17mm, above 18mm, above 19mm, above 20mm, above 21mm, or above 22mm.

[0090] The liquid outlet of the liquid outlet tube 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 limitation and be connected to a tubular structure such as a silicone hose. In addition, if the flow rate of the culture medium in the liquid outlet tube body 7 is too large, the flow rate of the culture medium in the main tube body 6 will also be very large, and the sedimentation time of the microcarrier in the main tube body 6 will be too short to complete the sedimentation, and the microcarrier will be extracted by the extraction power device. In actual applications, the specific flow rate varies according to the density of the microtissue (such as microcarrier) and is adjusted accordingly.

[0091] At the same time, if the inner diameter of the liquid outlet tube 7 is equal to the inner diameter of the main tube 6, when the liquid is extracted through the liquid outlet of the liquid outlet tube 7, it will cause greater interference to the liquid in the main tube 6, that is, it will cause a greater flow, and the microcarriers will not be able to settle and will be extracted by the extraction power device.

[0092] Through this embodiment, the inner diameter of the liquid outlet tube body 7 is smaller than the inner diameter of the main tube body 6, which is conducive to the smooth progress of sedimentation. Optionally, the ratio of the inner diameter of the liquid outlet tube body 7 to the inner diameter of the main tube 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 length values ​​between any of the above values, or a range with any two of these values ​​as endpoints, preferably 1:8 to 1:4, 1:7 to 1:5, or 1:6.

[0093] Optionally, the top of the main body 6 has a tapered portion, and the inner diameter gradually decreases to adapt to the inner diameter of the liquid outlet pipe 7 for connection. Optionally, 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, and 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 that is suitable for the inner diameter of the liquid outlet 7.

[0094] In some embodiments, as shown in FIG. 3 , the bottom opening 8 of the liquid extraction tube 3 may also be connected to a tapered opening 9 , and the tapered opening 9 is located at the bottom of the main tube 6 .

[0095] The conical opening 9 can prevent microcarriers from gathering at the bottom of the main body 6. The size of the cross section of the conical opening 9 is continuously reduced along the vertical direction. As used herein, the term "conical" refers to a three-dimensional shape structure having a top surface and non-parallel side walls, which gradually taper to a bottom surface with a small but non-zero area. The conical structure can have a cross-sectional shape of a circle, a triangle, a square, a pentagon, a hexagon, etc. Exemplary conical structures can be cones, pyramids, etc. The liquid flow in the tank body 1 can disperse the microcarriers at the bottom of the conical opening 9 to prevent the microcarriers from gathering. At the same time, because the conical opening 9 has a small opening, 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 microcarriers and cells thereon being extracted. The conical opening 9 includes an outlet side connected to the main body 6 and a corresponding inlet side, and the culture medium suspension mixed with microcarriers enters and passes through the conical 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 pipe body 6, and the inner diameter of the inlet side is smaller than the inner diameter of the main pipe body 6. Optionally, the ratio of the inner diameter of the inlet side of the tapered opening 9 to the inner diameter of 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, 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 length values ​​between any of the above values, or ranges with any two of these values ​​as endpoints, preferably 1:2 to less than 1:1, 1:18 to 1:1.05, etc.

[0096] This embodiment can help prevent the microcarriers from being drawn out of the tank.

[0097] Optionally, since the cross-sectional size of the tapered opening 9 gradually decreases, after the reinforcement component is placed in the main tube 6 , the tapered opening 9 also plays a role in fixing the reinforcement component.

[0098] In some embodiments, as shown in FIG4 , the liquid extraction tube 3 may further include an enhanced flow guide assembly (also referred to herein as an enhanced assembly). The enhanced assembly includes a vertical blade fixing rod 10, a plurality of blades 11 fixed to the blade fixing rod, an optional assembly fixing rod 12 at the bottom end of the blade fixing rod, and a handle 13.

[0099] The blade fixing rod 10 is detachably fixed in the liquid pumping tube 3 , so when the blade fixing rod 10 needs to be replaced or maintained, the entire liquid pumping tube 3 does not need to be replaced.

[0100] The plurality of blades 11 can block the microcarriers and the cells thereon, and help the microcarriers and the cells thereon to settle.

[0101] By this embodiment, the blades 11 are useful in assisting the sedimentation of the microcarriers and the cells thereon.

[0102] In some embodiments, 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 by means of a bottom connection. Optionally, the bottom end of the blade fixing rod 10 is connected to the bottom end of the main body 6. Specifically, as shown in Figures 4A and 4B, a hanging structure 14 is made on the bottom opening 8 of the liquid extraction tube 3, and the enhanced component can be detachably fixed in the liquid extraction tube 3 by hanging the component fixing rod 12 on the hanging structure 14. Optionally, the handle 13 of the enhanced component is designed below the component fixing rod 12, so that the handle 13 is located outside the lower opening of the liquid extraction tube, so as to facilitate the installation and removal of the enhanced component by holding the handle 13. Among them, Figure 4A is an explosion schematic diagram, and Figure 4B shows a schematic diagram of the installation of the enhanced diversion component and the liquid extraction tube. Optionally, one or more, for example, 1, 2, 3, 4 or more component fixing rods 12 are arranged on the bottom end or the outer circumferential surface near the bottom end of the blade fixing rod 10. For example, the component fixing rod is in the shape of a round rod. The shape of the component fixing rod is not particularly limited and can be a shape known in the art as long as it can play a fixing role. The multiple component fixing rods can be evenly distributed along the bottom end or the outer circumferential surface near the bottom end of the blade fixing rod 10. When the reinforcement component is placed in the main pipe body 6 and the conical opening 9 is installed and connected, the component fixing rod contacts the conical opening 9 to fix the reinforcement component in the liquid extraction tube 3. Optionally, the handle 13 of the reinforcement component is designed below the component fixing rod 12, so that the handle 13 is located outside the lower opening of the liquid extraction tube, so as to facilitate the installation and removal of the reinforcement component by holding the handle 13.

[0103] Optionally, the length of the blade fixing rod 10 is such that the top end of the blade fixing rod abuts against the top of the main pipe body 6 , and the bottom end of the blade fixing rod is connected to the bottom end of the main pipe body or abuts against the conical opening 9 , thereby fixing the reinforcement component in the liquid extraction pipe 3 .

[0104] 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%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less of the length of the main body 6, and the length between any of the above values, or the range with any two of the above values ​​as endpoints.

[0105] Optionally, the length of the blade fixing rod 10 may be 50% of the length of the main tube 6 .

[0106] Optionally, the length of the blade fixing rod 10 can be equal to the length of the main body 6, or 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% of the length of the main body 6, or a range with any two of the above values ​​as endpoints, for example, 90%-95%.

[0107] The longer the blade fixing rod 10 is, the more conducive it is to the settlement.

[0108] The shorter the blade fixing rod 10 is, the more it helps to reduce its influence on the cells in the bioreactor.

[0109] In some embodiments, the blade 11 is perpendicular to the blade fixing rod 10 .

[0110] Through this embodiment, the blocking effect of the blades 11 can be maximized, which helps to make sedimentation efficient.

[0111] In some embodiments, the angle between the blade 11 and the vertical direction of the blade fixing rod 10 is an acute angle, for example, the angle can be 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, etc., as well as the length between any of the above values, or the range with any two of the above values ​​as endpoints, preferably 30° to 60°, for example, 30°, 45° or 60°. For example, as shown in Figures 5C to 5E, 5A is a longitudinal cross-sectional view of the liquid extraction tube enhancement assembly; 5B is a top view of the liquid extraction tube enhancement assembly; the angle between the blade and the vertical direction of the blade fixing rod in 5C is 30°; the angle between the blade and the vertical direction of the blade fixing rod in 5D is 45°; the angle between the blade and the vertical direction of the blade fixing rod in 5E is 60°.

[0112] Through this embodiment, the blades can also play a blocking role and help the sedimentation process.

[0113] According to this embodiment, compared with the case where the blade 11 is perpendicular to the blade fixing rod 10 , the impact force of the collision between the blade 11 and the cell can be weakened, thereby facilitating the protection of the cell.

[0114] Optionally, as shown in FIG. 5C , the included angle between the blade 11 and the vertical direction (axial center line) of the blade fixing rod 10 is 30°.

[0115] Optionally, as shown in FIG. 5D , the included angle between the blade 11 and the vertical direction (axial center line) of the blade fixing rod 10 is 45°.

[0116] Optionally, as shown in FIG. 5E , the included angle between the blade 11 and the vertical direction (axial center line) of the blade fixing rod 10 is 60°.

[0117] The included angle between the blade 11 and the blade fixing rod 10 in the vertical direction can be changed according to specific needs.

[0118] In some embodiments, the blades 11 are rectangular, circular, elliptical or spiral.

[0119] Optionally, the vertical angle between the blade 11 and the blade fixing rod 10 can be changed according to specific needs.

[0120] Optionally, the blade 11 may be arranged perpendicular to the blade fixing rod 10 .

[0121] Optionally, the blade 11 may be rectangular, trapezoidal, polygonal, triangular, circular, elliptical, conical, fan-shaped, umbrella-shaped, curved, etc. Optionally, the blade 11 is spiral, that is, a plurality of blades together form a spiral blade with the blade fixing rod 10 as the axis; or, a plurality of blades are distributed in a spiral around the blade fixing rod 10. Such a distribution method can more effectively exert the blocking effect of the blade 11.

[0122] Optionally, the size of the blade 11 is not particularly limited. The maximum width of the liquid extraction tube enhancement component is smaller than the inner diameter of the main body, so that it can be set in the main body. Preferably, the maximum width of the liquid extraction tube enhancement component is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% of the inner diameter of the main body, 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 smaller the difference between the width of the liquid extraction tube enhancement component and the inner diameter of the main body, the smaller the distance between the edge of the blade and the wall of the main body, and the better it can block the microcarrier.

[0123] Optionally, the included angle between the blade 11 and the vertical direction of the blade fixing rod 10 may be an acute angle, a right angle or an obtuse angle.

[0124] Optionally, the angles between the plurality of blades 11 and the blade fixing rod 10 may be equal or different.

[0125] Optionally, the plurality of blades 11 have the same or different shapes. Preferably, the plurality of blades 11 have the same shape.

[0126] Optionally, in some embodiments, the plurality of blades 11 are evenly distributed on the blade fixing rod 10. That is, the plurality of blades 11 are evenly spaced in the vertical direction.

[0127] Optionally, in some embodiments, the plurality of blades 11 are non-uniformly distributed on the blade fixing rod 10. That is, the plurality of blades 11 are not spaced evenly apart in the vertical direction.

[0128] Optionally, along the vertical direction, the density of the blades 11 on the blade fixing rod 10 gradually increases. That is, along the vertical direction, the spacing between adjacent blades gradually decreases.

[0129] Optionally, the blade 11 is connected and fixed to the blade fixing rod 10 through a fixing member, for example, a fixing member installed on the edge of the blade is connected and fixed to the blade fixing rod 10. Alternatively, optionally, the blade fixing rod 10 passes through the body of the blade 11, for example, through the body of the blade 11 in a perforated manner, so as to fix 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 from the geometric center of the blade 11 body shape, such as the center of a circular or elliptical blade, and the top cone point of a conical or umbrella-shaped blade.

[0130] According to this embodiment, since sedimentation mainly occurs at the bottom of the liquid extraction tube 3, it helps to improve the sedimentation effect.

[0131] In some embodiments, the multiple blades 11 are divided into multiple blade groups, each blade group includes multiple rectangular blades 11, and the multiple rectangular blades 11 of each blade group are located on the same horizontal plane of the blade fixing rod 10 and connected to the blade fixing rod 10 through the blade root (fixing piece).

[0132] Optionally, each blade group may include the same number or different numbers of blades 11 .

[0133] Optionally, the shape of the blades 11 in the blade group may be rectangular, trapezoidal, polygonal, triangular, circular, elliptical, fan-shaped, etc., or may be conical, umbrella-shaped, curved, etc.

[0134] Optionally, the blade group may include 2, 3, 4 or more blades 11. Preferably, the blades 11 in each blade group 11 are evenly distributed around the blade fixing rod 10, for example, when the blade group includes 2 blades 11, the angle between the blades 11 is 180°, and when the blade group includes 3 blades 11, the angle between the blades 11 is 120°.

[0135] Optionally, the spacings between the plurality of blade groups in the vertical direction are equal; or the spacings between adjacent blade groups in the vertical direction gradually decrease.

[0136] Optionally, as shown in FIG. 5B , the blades 11 of two upper and lower adjacent blade groups are staggered in the circumferential direction.

[0137] Optionally, the blades 11 of two upper and lower adjacent blade groups maintain consistent positions in the circumferential direction.

[0138] Optionally, the plurality of blades 11 have the same or different shapes, preferably the plurality of blades 11 have the same shape.

[0139] As shown in FIG. 1 and FIG. 2 , this embodiment further provides a bioreactor, which includes the above-mentioned liquid pumping device.

[0140] By utilizing this bioreactor, during the process of extracting the culture medium liquid and separating the culture medium from the microcarriers, since the use of the traditional microporous filtration method is avoided, blockage can be effectively avoided, thereby enabling continuous extraction of the culture medium liquid for a long period of time; at the same time, since the liquid extraction tube assembly is inserted into the tank body 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, thereby avoiding a greater impact on the more fragile cells.

[0141] In some embodiments, the bioreactor also includes a top cover 2, a tank body 1, a stirring assembly and a liquid inlet pipe 18, the stirring assembly includes: a stirring shaft 4, the stirring shaft 4 is fixed on the top cover 2 through a bearing seat; and a stirring paddle 17, which 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.

[0142] Optionally, the stirring assembly may also be arranged at the bottom of the tank body 1, in which case the liquid extraction pipe assembly and the liquid inlet pipe 18 are arranged to avoid each other.

[0143] As shown in FIG1 , the liquid extraction pipe assembly, the liquid inlet pipe 18 and the stirring assembly are all arranged at intervals, that is, the liquid extraction pipe assembly, the liquid inlet pipe 18 and the stirring assembly are far away from each other, so the liquid extraction and liquid inlet processes of the bioreactor do not interfere with each other and will not affect the normal operation of the stirring assembly.

[0144] Optionally, the stirring assembly is arranged at the center of the tank body 1, and the liquid extraction pipe assembly and the liquid inlet pipe 18 are arranged to avoid the stirring assembly.

[0145] The stirring shaft 4 is fixed on the top cover 2 through a bearing seat, so the stirring shaft 4 can rotate to achieve stirring.

[0146] The stirring paddle 17 is fixed on the stirring shaft 4, which helps to further improve the stirring efficiency.

[0147] Through this implementation, the liquid extraction process of the bioreactor will not affect the normal operation of the stirring component.

[0148] 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 extraction pipe 3, which is conducive to the replacement of fresh culture medium and culture medium with consumed nutrients.

[0149] If the lower end of the liquid inlet pipe 18 is at the same or lower level than the bottom opening 8 of the liquid extraction pipe 3, the fresh culture medium will enter the tank through the lower end of the liquid inlet pipe 18 and will be extracted by the liquid extraction pipe. By setting the lower end of the liquid inlet pipe 18 at a higher level, the culture medium enters the tank above the bottom opening 8 of the liquid extraction pipe 3, and has enough opportunities to be stirred by the stirring shaft and mixed with the culture medium consumed by nutrients, thereby achieving the purpose of continuously perfusing the tank with fresh culture medium.

[0150] The present embodiment also provides a method for extracting liquid using the above-mentioned liquid extraction device, and 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 tank body 1 of the bioreactor; liquid is extracted by the liquid extraction device, and during the liquid extraction process, the microcarriers entering the liquid extraction tube assembly settle in the liquid extraction tube assembly and eventually detach from the liquid extraction tube assembly to remain in the tank body 1, and the culture medium liquid leaves the bioreactor from the top of the liquid extraction tube assembly.

[0151] By utilizing this extraction method, the extraction of the culture medium liquid and the separation process of the culture medium and the microcarrier can effectively avoid blockage due to avoiding the use of traditional microporous filtration methods, thereby allowing the culture medium liquid to be extracted for a long time; at the same time, since the extraction tube assembly is inserted into the tank body 1 of the bioreactor, the cells on the microcarrier entering or leaving the extraction tube assembly will not cause the cells on the microcarrier to leave the culture environment in the bioreactor tank, thereby avoiding a greater impact on the more fragile cells.

[0152] In the method disclosed in the present invention, there is no particular restriction on the pumping rate, and the pumping rate can be adjusted according to the size of the tank and the size of the corresponding pumping device. Optionally, the pumping rate can meet the liquid replacement demand of the tank. For example, for a 5L tank, the amount of liquid replacement every 24 hours reaches more than 4L, that is, generally speaking, more than 80% of the volume of the tank, such as more than 85%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, thereby calculating the pumping rate that meets the liquid replacement demand. For each volume of the tank, the liquid replacement demand is known in the art and can be easily determined by a person skilled in the art. The faster the pumping rate, the faster the culture medium consumed by nutrients is discharged, and the faster the fresh culture medium can be filled, which is conducive to the growth of cells. Optionally, the pumping rate can be greater than the pumping rate calculated above according to actual needs. For example, based on the pumping rate V0 calculated to meet the requirement of 100% of the tank volume during 24-hour liquid replacement, the pumping 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, or 10 times that of V0.

[0153] Optionally, the pumping rate may be smaller than the calculated pumping rate according to actual needs.

[0154] Optionally, the pumping rate is up to 4 mL / min, up to 5 mL / min, up to 6 mL / min, up to 7 mL / min, up to 8 mL / min, up to 9 mL / min, up to 10 mL / min, up to 11 mL / min, up to 12 mL / min, up to 13 mL / min, up to 14 mL / min, up to 15 mL / min, up to 16 mL / min, up to 17 mL / min, up to 18 mL / min, up to 19 mL / min, up to 20 mL / min, up to 21 mL / 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, 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 to 50mL / min, up to 60mL / min, up to 70mL / min, up to 80mL / min, up to 90mL / min, up to 100mL / min.

[0155] In some embodiments, the microcarrier enters the liquid extraction tube 3 through the tapered opening 9 , settles in the main tube 6 of the liquid extraction tube 3 , and then leaves the liquid extraction tube 3 through the tapered opening 9 .

[0156] The conical opening 9 can prevent the microcarriers from gathering at the bottom of the main tube 6. The cross-sectional size of the conical opening 9 is continuously reduced along the vertical direction. The liquid flow in the tank 1 can disperse the microcarriers at the bottom of the conical opening 9 to prevent the microcarriers from gathering. At the same time, because the conical opening 9 is small, it can prevent the main tube 6 from entering too much culture medium suspension mixed with microcarriers at one time, further preventing the possibility of the microcarriers and the cells thereon from being extracted.

[0157] This embodiment can help prevent the microcarriers from being drawn out of the tank.

[0158] In some embodiments, the blades 11 prevent the microcarriers from moving upward in the main body 6 of the liquid suction tube 3 .

[0159] The plurality of blades 11 can block the microcarriers and the cells thereon, and help the microcarriers and the cells thereon to settle.

[0160] The blade 11 is fixed on the blade fixing rod 10 .

[0161] By this embodiment, the blades 11 are useful in assisting the sedimentation of the microcarriers and the cells thereon.

[0162] Example

[0163] The technical scheme of the present invention is further described in detail below by examples and in conjunction with the accompanying drawings. The examples given are only for illustrating the present invention, but not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0164] Example 1

[0165] This embodiment specifically exemplifies 7 types of liquid extraction devices, and the actual types are shown in FIG7 . The specific size parameters are shown in the following table. These geometric dimensions are related to the size of the bioreactor tank and are not limited to specific parameters.

[0166] Table 1: Specific dimensional parameters of the pumping device and reinforcement components

[0167] Note: When the inner diameter of the bottom opening in the table is equal to the inner diameter of the main body, the bottom end of the main body is not connected to the tapered opening; when the inner diameter of the bottom opening is smaller than the inner diameter of the main body, the bottom end of the main body is connected to a tapered opening, and the inner diameter of the bottom opening refers to the inner diameter of the tapered opening.

[0168] Example 2

[0169] This example uses a bioreactor of the present invention to perform a liquid extraction experiment of a microcarrier suspension.

[0170] The pumping device used in the bioreactor is the 1#, 3# and 5# pumping devices in Example 1, that is, 1# uses a pumping device alone, 3# uses a long reinforcement component (250mm) in combination, and 5# uses a short reinforcement component (152mm) in combination. The reinforcement component adopts a rectangular blade, and the angle between the blade 11 and the blade fixing rod 10 is 60°. This embodiment mainly tests whether it is possible to extract a single liquid without microcarrier clogging or being pumped out under a low-concentration porous microcarrier system. These geometric dimensions are related to the size of the bioreactor tank. The dimensions designed in this embodiment match the stirred bioreactor (model: FTVS05, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the embodiment, and are not limited as specific parameters.

[0171] The specific experimental operations are as follows.

[0172] 1. Add the dried microcarriers into sterile water at a concentration of 1g / L and soak them for swelling for 24 hours.

[0173] 2. Transfer 5 L of swollen microcarrier suspension into tank 1 of the bioreactor through the feed tube.

[0174] 3. Start the stirring assembly of the bioreactor and stir at 50 rpm for 2 hours to ensure that the microcarriers are fully dispersed and suspended in the tank.

[0175] 4. The liquid outlet of the liquid outlet tube body 7 is connected to the peristaltic pump through a silicone hose with an inner diameter of 3.1 mm, and the other end of the hose is connected to a liquid receiving container, such as a beaker.

[0176] 5. Start the peristaltic pump while the bioreactor is stirring at 50 rpm. Set the peristaltic pump speed to 6-14 rpm, and the corresponding pumping rate is 2.8-6.58 mL / min. Continue pumping for 8 hours.

[0177] 6. Observe whether the liquid in the hose flows smoothly to determine whether the pumping device is blocked. If blockage occurs, the liquid extracted from the pumping device will gradually decrease, and the hose will not be able to be filled, resulting in more cavitation in the hose.

[0178] 7. Observe whether there are microcarriers in the liquid receiving container to determine whether the liquid extraction device can effectively separate the microcarriers and the liquid. If there are microcarriers in the liquid receiving container, it means that the liquid extraction device cannot separate the microcarriers and the liquid, and thus the purpose of the present invention cannot be achieved.

[0179] 8. As shown in Table 1, the pumping 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 pumping rate is 2.8mL / min, which can complete the liquid replacement of about 4L within 24 hours a day, basically meeting the liquid replacement needs of a 5L bioreactor. The results further show that the use of an enhanced component can further prevent the microcarriers from being extracted, and the closer the pumping rate and the length of the enhanced component are to the length of the main body, the better the effect. The short enhanced component can extract liquid faster than the pumping device without an enhanced component without discharging liquid, specifically it can be increased to 3.29mL / min, and the long enhanced component can further increase the pumping rate to 4.7mL / min. The faster the pumping rate, the faster the cells can discharge the culture medium consumed by nutrients, and then the faster the fresh culture medium can be filled, which is beneficial to the growth of cells. The experimental results also show that regardless of whether the enhanced component is used, no blockage occurs during the entire pumping process.

[0180] Table 2: Microcarrier separation and extraction effect of the extraction device and the enhanced component

[0181] Example 3

[0182] This embodiment uses a bioreactor of the present invention to carry out a liquid extraction experiment of a microcarrier suspension. The liquid extraction device used in the bioreactor is the 2# to 7# device in Example 1. The main purpose is to test whether a single liquid can be extracted without microcarrier clogging or being pumped out under a higher concentration of solid sphere microcarrier system. These geometric dimensions are related to the size of the bioreactor tank. The dimensions designed in this embodiment match the stirred bioreactor (model: FTVS05, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the embodiment and are not limited as specific parameters.

[0183] The specific experimental operations are as follows.

[0184] 1. The dried microcarriers were added to sterile phosphate buffer (pH 7.4) at a concentration of 8.3 g / L and soaked for 24 hours, and then sterilized by high pressure at 121°C for 40 minutes.

[0185] 2. Transfer 3L of swollen microcarrier suspension into tank 1 of the bioreactor through the feeding tube.

[0186] 3. Start the stirring assembly of the bioreactor and stir at 60 rpm for 2 hours to ensure that the microcarriers are fully dispersed and suspended in the tank.

[0187] 4. Connect the liquid outlet of the liquid outlet tube body 7 of the liquid extraction device to the peristaltic pump through a silicone hose with an inner diameter of 3.1 mm, and connect the other end of the hose to a liquid receiving container, such as a beaker.

[0188] 5. While the bioreactor is stirring at 60 rpm, start the peristaltic pump and set the peristaltic pump rate to 8 rpm, corresponding to a pumping rate of 3.52 mL / min, or about 5 L / day. Continue pumping and at the same time add phosphate buffer to the bioreactor tank at the same liquid inlet rate to maintain a constant concentration of microcarriers in the tube.

[0189] 6. Take 200 μL of solution from the liquid receiving container at 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 19 hours, 24 hours and 48 hours after the start of liquid extraction, and observe under a microscope whether there are microcarriers to determine whether the liquid extraction device can effectively separate the microcarriers and liquid. If there are microcarriers in the liquid receiving container, it means that the liquid extraction device cannot separate the microcarriers and liquid, and thus the purpose of the present invention cannot be achieved.

[0190] 7. When testing the 2# pumping device, 3# pumping device and 4# pumping device, the peristaltic pump rate was further increased to 16rpm and 24rpm, and the corresponding pumping rates were 7.04mL / min and 10.56mL / min, i.e., about 10L / day and 15L / day, respectively. The pumping was continued, and at the same time, phosphate buffer was added to the bioreactor tank at the same liquid inlet rate to maintain the concentration of microcarriers in the tube body unchanged. 180 minutes after the start of the pumping, 200μL of solution was taken from the ground liquid receiving container and observed under a microscope to see if there were microcarriers to determine whether the pumping device effectively separated the microcarriers and the liquid. If there were microcarriers in the liquid receiving container, it means that the pumping device cannot separate the microcarriers and the liquid, and thus the purpose of the present invention cannot be achieved.

[0191] 8. Analysis of results: Figure 8 shows that there are a large number of microcarriers in the liquid extracted using an ordinary discharge pipeline. Figures 9 and 10 show that a small amount of microcarriers were pumped out of the 5# and 6# devices at 48 hours, and a small amount of microcarriers were pumped out of the 7# device at 19 hours, while no carriers were pumped out of the other devices. The results show that whether it is a wide mouth or a conical mouth (2# vs. 3#), a rectangular blade or a circular blade (2# vs. 4#), it can prevent the microcarriers from being pumped out. In addition, longer reinforcement components (3# vs. 5#) are more effective than short reinforcement components, while the angle of the rectangular blade has no significant effect on the effect (5# vs. 6#). However, the use of 90° circular blades and the use of the holes on the blades to extract liquid increases the probability of microcarriers being pumped out, and microcarriers begin to appear at 19 hours (7#). Figure 11 shows that no microcarriers were pumped out in devices 2# and 3# at pumping rates of 3.52 mL / min, 7.04 mL / min, and 10.56 mL / min, while no microcarriers were pumped out in device 4# at a rate of 3.52 mL / min, but a large number of microcarriers were pumped out at pumping rates of 7.04 mL / min and 10.56 mL / min. Therefore, devices 2# and 3# are more suitable for high-rate pumping.

[0192] Example 4

[0193] 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# pumping device in Example 1, i.e., the pumping device containing an enhanced component, the blades of the enhanced component are rectangular blades with an angle of 60°, the length of the enhanced component is 250 mm, and the length of the main body is 255 mm. The specific operation is as follows.

[0194] 1. Install the 3# liquid extraction device in Example 1 in the tank of a stirred bioreactor. The tank is a bioreactor tank with a maximum working volume of 5L, and its glass tank body is 342mm high and 170mm in diameter. A thermoplastic tube with an inner diameter of 3.1mm is installed at the liquid outlet of the liquid extraction tube 3 and the liquid inlet tube 18, respectively, and the other ends of the two thermoplastic tubes are sealed with a welding sealer. The tank is sterilized in the same way as a conventional bioreactor tank, i.e., autoclaved at 121°C for 60min.

[0195] 2. After sterilization, connect the bioreactor tank to the bioreactor control host.

[0196] 3. In a biosafety cabinet, mix 4 g of microcarriers and 1 × 10 8 Mix 100 MSCs with 2 L of culture medium in a sterile storage bag.

[0197] 4. After aseptically welding the thermoplastic tube of the sterile liquid storage bag to the thermoplastic tube of the liquid inlet pipe 18 of the bioreactor, a peristaltic pump is used to pump the culture medium suspension of the microcarriers and cells into the bioreactor tank.

[0198] 5. After all the culture medium suspension of microcarriers and cells has entered the tank 1, inject 5L of fresh culture medium into the sterile liquid storage bag. This bag will be used as a feed bag for standby use.

[0199] 6. Start the stirring assembly of the bioreactor and stir at 35 rpm. At the same time, start the temperature, pH and dissolved oxygen control of the bioreactor, control the temperature at 37°C, pH at 7.0 and dissolved oxygen at more than 50%, and carry out cell culture. This is defined as day 0.

[0200] 7. On day 1, 1 L of fresh culture medium is pumped into tank 1 through inlet tube 18.

[0201] 8. On the second day, the thermoplastic tube of a new empty sterile liquid storage bag (used as a container for receiving waste culture fluid, hereinafter referred to as the waste liquid bag) was aseptically welded to the thermoplastic tube on the outlet of the liquid extraction device. The perfusion program was started, that is, the peristaltic pump connected to the outlet of the liquid extraction tube was started to extract liquid at 2.82 mL / min, and the peristaltic pump at the inlet was started at the same time to supplement the nutrient-rich culture medium from the feed bag to the tank at 2.82 mL / min.

[0202] 9. Continue culturing until the 5th day.

[0203] 10. Take the microcarriers containing cells from the bioreactor as samples every day for cell number detection and glucose content detection, where the change in glucose content represents the change in nutrient content.

[0204] 11. Results: Figure 12 shows the changes in glucose content and cell number. Generally, as the number of cells increases, the nutrients in the culture medium are consumed, represented by glucose, and its content decreases accordingly. However, the results show that under the condition of a perfusion rate of 6rpm, in the first 4 days of culture, as the number of cells increases, the glucose content does not decrease significantly (only from 33mM to 31.4mM). It can be seen that the culture fluid consumed by nutrients can be extracted through this pumping device and then supplemented with fresh culture medium through the liquid inlet tube to maintain sufficient nutrients in the culture system. From the 4th day to the 5th day, the glucose content dropped significantly to 23.7mM, and the cells had an explosive growth. The reason why the perfusion did not maintain the nutrient content at this time may be that the pumping rate and liquid inlet rate of 2.82mL / min were too slow, and the speed of fresh culture medium replenishment could not keep up with the speed of cell consumption of nutrients. Figure 13 shows the liquid discharged from the outlet of the liquid pumping device in the waste liquid bag. During the three-day perfusion process, no microcarriers were observed under the microscope at the three observation time points. Moreover, on the 5th day of culture (the 3rd day of perfusion), FIG. 14 shows that the pipeline was still filled with liquid without cavitation or foam, indicating that the pumping device was not blocked.

[0205] Example 5

[0206] 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# pumping device in Example 1, i.e., the pumping device containing the enhanced component, the blades of the enhanced component are rectangular blades with an angle of 60°, the length of the enhanced component is 162 mm, and the length of the main body is 165 mm. The specific operation is as follows.

[0207] 1. Install the 8# pumping device in Example 1 in the bioreactor tank 1 of the present invention. In the specific application of the present embodiment, the tank 1 is designed as a bioreactor tank with a maximum working volume of 2L, and the glass tank body has a height of 219mm and a diameter of 140mm.

[0208] 2. Add 10 g of microcarriers and 1 L of PBS into the tank.

[0209] 3. A thermoplastic tube with an inner diameter of 3.1 mm is installed at the liquid outlet of the liquid extraction pipe 3 and the liquid inlet pipe 18 of the liquid extraction device, and the other ends of the two thermoplastic tubes are sealed with a welding sealing machine.

[0210] 4. Sterilize the tank in the same manner as a conventional bioreactor, i.e., autoclave at 121°C for 60 minutes.

[0211] 5. After sterilization, connect the bioreactor tank to the bioreactor control host.

[0212] 6. The microcarriers naturally settle without stirring. After aseptically welding the thermoplastic tube on the sterile waste liquid bottle to the thermoplastic tube on the outlet of the pumping device of the bioreactor, use a peristaltic pump to discharge the PBS supernatant from the glass jar, pump in 1L of culture medium, stir evenly, stop stirring again to allow the microcarriers to settle naturally, and pump out the basal culture medium supernatant again. At this time, the volume of the microcarrier and M199 suspension is about 300mL.

[0213] 7. 8.7×10 7 VERO cells were mixed with 1 L of culture medium in a sterile feed bottle.

[0214] 8. After aseptically welding the thermoplastic tube of the sterile feeding bottle to 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.

[0215] 9. After all the culture medium suspension of microcarriers and cells has entered tank 1, inject 14L of fresh complete culture medium into the sterile feeding bottle for standby use.

[0216] 10. Start the stirring assembly of the bioreactor at 35 rpm, and start the temperature, pH and dissolved oxygen control of the bioreactor at the same time, control the temperature at 37°C, pH at 7.0 and dissolved oxygen at more than 50% to culture cells. This is defined as day 0.

[0217] 11. Starting from the third day, the perfusion program is started, that is, the peristaltic pump connected to the liquid outlet of the liquid extraction tube is started to extract liquid into the waste liquid bottle at 3rpm (i.e. 1.41mL / min), and the peristaltic pump at the liquid inlet is started at the same rate to replenish the nutrient-rich culture medium from the feed bottle into the tank. This speed can achieve a 2L liquid change per day, that is, a culture medium replacement of one tank volume per day.

[0218] 12. Starting from the 4th day, the perfusion program is started, that is, the peristaltic pump connected to the liquid outlet of the liquid extraction tube is started to extract liquid into the waste liquid bottle at 6rpm (i.e. 2.82mL / min), and the peristaltic pump at the liquid inlet is started at the same rate to replenish the nutrient-rich culture medium from the feed bottle into the tank. This speed can achieve 4L of liquid replacement per day, that is, the culture medium of 2 tank volumes can be replaced per day.

[0219] 13. Continue culturing until the 7th day.

[0220] 14. Take the microcarriers containing cells from the bioreactor as samples every day for cell number detection and glucose content detection, where the change in glucose content represents the change in nutrient content.

[0221] 15. Results: Figure 15 shows the changes in glucose content and cell number. From day 0 to day 3, without perfusion, as the number of cells increased, the nutrients in the culture medium were consumed, represented by glucose, and its content decreased accordingly. After perfusion began on day 3, although the cells had an explosive growth, the glucose did not drop drastically, but remained at a certain concentration (above 16 mm). It can be seen that the culture medium that has consumed nutrients can be extracted through this pumping device and then supplemented with fresh culture medium through the liquid inlet tube to maintain sufficient nutrients in the culture system. The continuous increase in the number of cells also proves that the microcarriers containing the cells have not been discharged from the tank, otherwise, as the perfusion is pumped out of the tank, the number of cells will continue to decrease. Figure 16 shows that the liquid discharged from the outlet of the pumping device is clear waste liquid, and no microcarriers are discharged.

[0222] Example 6

[0223] In this embodiment, 8 types of liquid extraction devices are specifically shown, and the actual types are shown in Figure 17. The specific size parameters are shown in the following table. These geometric dimensions are related to the size of the bioreactor tank and are not limited to specific parameters.

[0224] Table 3: Specific dimensional parameters of the pumping device and reinforcement components

[0225] Note: When the inner diameter of the bottom opening in the table is smaller than the inner diameter of the main body, it is because there is a tapered opening connected to the bottom end of the main body. In this case, the inner diameter of the bottom opening refers to the inner diameter of the tapered opening.

[0226] Example 7

[0227] This example uses a bioreactor of the present invention to perform a liquid extraction experiment of a microcarrier suspension.

[0228] The pumping device used in the bioreactor is the 9# and 10# pumping devices in Example 6, that is, the 9# pumping device uses an elliptical reinforcement component (156mm, as shown in Figure 18), and the 10# uses a spiral blade reinforcement component (156mm, as shown in Figure 19). This embodiment mainly tests which of the elliptical reinforcement components and the spiral blade reinforcement components under the 2L reactor microcarrier system culture is better. These geometric dimensions are related to the size of the bioreactor tank. The size designed in this embodiment matches the stirred bioreactor used in the embodiment and is not limited as a specific parameter.

[0229] Some of the equipment and reagents used in the following examples are as follows:

[0230] Commercially available microcarriers, such as Cytodex or 3D porous microcarriers. DMEM medium is a product of Shanghai Yuanpei Biotechnology Co., Ltd., item number: L120KJ, which is a conventional commercial product.

[0231] Fetal bovine serum is a product of Vicente Biotechnology Co., Ltd., item number: 086-150, and is a conventional commercially available product.

[0232] PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercially available product.

[0233] Recombinant trypsin digestion solution is a product of Lanzhou Minhai Company, item number: S342KJ, which is a conventional commercially available product.

[0234] Vero cells are African green monkey kidney cells, which are common cells in the art.

[0235] The specific experimental operations are as follows.

[0236] 1. Preparation of microcarriers: Weigh 4.5g of microcarriers, add 1000ml of PBS solution, and sterilize at 121℃ for 30min. After the microcarriers are cooled, add 1.0LDMEM culture medium, stir thoroughly, and then settle the microcarriers and discard the supernatant.

[0237] 2. Cell inoculation: Digest freshly cultured Vero cells with recombinant protease digestion solution, resuspend the cells in DMEM medium containing 10% newborn calf serum, inoculate the cells at a density of 300,000-500,000 / ml, the culture volume is 1.5 L, adjust the bioreactor parameters, and start culturing the cells.

[0238] 3. After 24 hours, start the perfusion fluid replacement procedure. Stir the culture at a speed of 40 rpm to ensure that the microcarriers are fully dispersed and suspended in the tank.

[0239] 4. The liquid outlet of the liquid outlet tube body 7 is connected to the peristaltic pump through a silicone hose with an inner diameter of 3.1 mm, and the other end of the hose is connected to a liquid storage container, such as a 10L glass liquid storage bottle.

[0240] 5. Start the peristaltic pump while the bioreactor is stirring at 40 rpm. The peristaltic pump rate can be adjusted. Pump liquid continuously during the culture process.

[0241] 6. Observe whether the liquid in the hose flows smoothly or whether the liquid discharge speed is consistent to determine whether the pumping device is blocked. If blockage occurs, the speed of extracting liquid from the pumping device gradually slows down or the liquid in the pumping tube gradually decreases, making it impossible to fill the hose and forming more cavitations in the hose.

[0242] 7. Observe whether there are microcarriers in the liquid storage container to determine whether the liquid extraction device can effectively separate the microcarriers and liquid. If there are microcarriers in the liquid storage container, it means that the liquid extraction device cannot separate the microcarriers and liquid, and thus the purpose of the present invention cannot be achieved.

[0243] 8. On the premise of meeting 6 and 7, record the maximum extraction speed of the peristaltic pump of the elliptical reinforcement component (156mm) and the spiral blade reinforcement component (156mm) during the culture process every day.

[0244] 9. As shown in Figure 26, the oval 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 reaches 7.05mL / min, and there is no blockage or microcarrier extraction. It can complete about 10L of liquid replacement within 24 hours a day, which fully meets the liquid replacement needs of a 2L bioreactor. As shown in Figure 27, the extraction rate of the spiral reinforcement component is as high as 7.05mL / min at the beginning, but as the culture progresses in the later stage, the extraction rate decreases to 2.35mL / min, indicating that the spiral reinforcement component will accumulate microcarriers; the faster the liquid extraction rate, the faster the cells can discharge the culture medium consumed by nutrients, and then the faster the fresh culture medium can be injected, which is beneficial to the growth of cells.

[0245] Table 4: Microcarrier separation and extraction effect of the extraction device and the enhanced component

[0246] Example 8

[0247] This example uses a bioreactor of the present invention to perform a liquid extraction experiment of a microcarrier suspension.

[0248] The pumping device used in this bioreactor is the 11# pumping device in Example 6, i.e., the 11# pumping device uses an elliptical reinforcement assembly (266 mm, as shown in Figure 20). This embodiment mainly tests the perfusion effect of the elliptical reinforcement assembly under the cultivation of the 5L reactor microcarrier system. These geometric dimensions are related to the bioreactor tank size. The dimensions designed in this embodiment match the stirred bioreactor (model: FTVS05, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the embodiment and are not limited as specific parameters.

[0249] Some of the equipment and reagents used in the following examples are as follows:

[0250] Commercially available microcarriers such as Cytodex or porous microcarriers.

[0251] DMEM culture medium is a product of Shanghai Yuanpei Biotechnology Co., Ltd., product number: L120KJ, and is a conventional commercially available product.

[0252] Fetal bovine serum is a product of Vicente Biotechnology Co., Ltd., item number: 086-150, and is a conventional commercially available product.

[0253] PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercially available product.

[0254] Recombinant trypsin digestion solution is a product of Lanzhou Minhai Company, item number: S342KJ, which is a conventional commercially available product.

[0255] Vero cells are African green monkey kidney cells, which are common cells in the art.

[0256] The specific experimental operations are as follows.

[0257] 1. Preparation of microcarriers: Weigh 9g of microcarriers, add 2000ml of PBS solution, and sterilize at 121℃ for 30min. After the microcarriers are cooled, add 2.0LDMEM culture medium, stir thoroughly, and then settle the microcarriers and discard the supernatant.

[0258] 2. Cell inoculation: Digest freshly cultured Vero cells with recombinant protease digestion solution, resuspend the cells in DMEM medium containing 10% newborn calf serum, inoculate the cells at a density of 300,000-500,000 / ml, the culture volume is 3L, adjust the bioreactor parameters, and start culturing the cells.

[0259] 3. After 24 hours, start the perfusion fluid replacement procedure. Stir the culture at a speed of 40 rpm to ensure that the microcarriers are fully dispersed and suspended in the tank.

[0260] 4. The liquid outlet of the liquid outlet tube body 7 is connected to the peristaltic pump through a silicone hose with an inner diameter of 3.1 mm, and the other end of the hose is connected to a liquid storage container, such as a 10L glass liquid storage bottle.

[0261] 5. Start the peristaltic pump while the bioreactor is stirring at 50 rpm. The peristaltic pump rate can be adjusted. Pump liquid continuously during the culture process.

[0262] 6. Observe whether the liquid in the hose flows smoothly or whether the liquid discharge speed is consistent to determine whether the pumping device is blocked. If blockage occurs, the speed of extracting liquid from the pumping device gradually slows down or the liquid in the pumping tube gradually decreases, making it impossible to fill the hose and forming more cavitations in the hose.

[0263] 7. Observe whether there are microcarriers in the liquid storage container to determine whether the liquid extraction device can effectively separate the microcarriers and liquid. If there are microcarriers in the liquid storage container, it means that the liquid extraction device cannot separate the microcarriers and liquid, and thus the purpose of the present invention cannot be achieved.

[0264] 8. On the premise of meeting 6 and 7, record the maximum extraction speed of the peristaltic pump of the elliptical reinforcement component (266mm) during the culture process every day.

[0265] 9. 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 reaches 7.99mL / min, and there is no blockage or microcarrier extraction. About 11.5L of liquid replacement can be completed within 24 hours a day, which basically meets the liquid replacement needs of a 5L bioreactor.

[0266] Table 5: Microcarrier separation and extraction effect of the extraction device and the enhanced component

[0267] Example 9

[0268] This example uses a bioreactor of the present invention to perform a liquid extraction experiment of a microcarrier suspension.

[0269] The pumping device used in this bioreactor is the 15# pumping device in Example 6, i.e., the 15# pumping device uses an elliptical reinforcement assembly (372 mm, as shown in Figure 24). This embodiment mainly tests the perfusion effect of the elliptical reinforcement assembly under the cultivation of the 15L reactor microcarrier system. These geometric dimensions are related to the bioreactor tank size, and the dimensions designed in this embodiment match the stirred bioreactor (model: FTVS15, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the embodiment, and are not limited as specific parameters.

[0270] Some of the equipment and reagents used in the following examples are as follows:

[0271] Commercially available microcarriers include Cytodex or 3D porous microcarriers. DMEM culture medium is a product of Shanghai Yuanpei Biotechnology Co., Ltd., item number: L120KJ, which is a conventional commercial product.

[0272] Fetal bovine serum is a product of Vicente Biotechnology Co., Ltd., item number: 086-150, and is a conventional commercially available product.

[0273] PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercially available product.

[0274] Recombinant trypsin digestion solution is a product of Lanzhou Minhai Company, item number: S342KJ, which is a conventional commercially available product.

[0275] Vero cells are African green monkey kidney cells, which are common cells in the art.

[0276] The specific experimental operations are as follows.

[0277] 1. Preparation of microcarriers: Weigh 36g of microcarriers, add 4000ml of PBS solution, and sterilize at 121℃ for 30min. After the microcarriers are cooled, add 8.0LDMEM culture medium, stir thoroughly, and then settle the microcarriers and discard the supernatant.

[0278] 2. Cell inoculation: Digest freshly cultured Vero cells with recombinant protease digestion solution, resuspend the cells in DMEM medium containing 10% newborn calf serum, inoculate the cells at a density of 300,000-500,000 / ml, the culture volume is 12L, adjust the bioreactor parameters, and start culturing the cells.

[0279] 3. After 24 hours, start the perfusion fluid replacement procedure. Stir the culture at a speed of 40 rpm to ensure that the microcarriers are fully dispersed and suspended in the tank.

[0280] 4. The liquid outlet of the liquid outlet tube body 7 is connected to the peristaltic pump through a silicone hose with an inner diameter of 4.8 mm, and the other end of the hose is connected to a liquid storage container, such as a 10L glass liquid storage bottle.

[0281] 5. Start the peristaltic pump while the bioreactor is stirring at 40 rpm. The peristaltic pump rate can be adjusted. Pump liquid continuously during the culture process.

[0282] 6. Observe whether the liquid in the hose flows smoothly or whether the liquid discharge speed is consistent to determine whether the pumping device is blocked. If blockage occurs, the speed of extracting liquid from the pumping device gradually slows down or the liquid in the pumping tube gradually decreases, making it impossible to fill the hose and forming more cavitations in the hose.

[0283] 7. Observe whether there are microcarriers in the liquid storage container to determine whether the liquid extraction device can effectively separate the microcarriers and liquid. If there are microcarriers in the liquid storage container, it means that the liquid extraction device cannot separate the microcarriers and liquid, and thus the purpose of the present invention cannot be achieved.

[0284] 8. On the premise of meeting 6 and 7, record the maximum extraction speed of the peristaltic pump of the elliptical reinforcement component (372 mm) during the culture process every day.

[0285] 9. 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.

[0286] Table 6: Microcarrier separation and extraction effect of the extraction device and the enhanced component

[0287] The microcarriers used in the above embodiments can be microcarriers well known in the art, including commercially available products, for example, solid sphere microcarriers Cytodex (Cytiva), 3D microslides (Beijing Huakan Biotechnology Co., Ltd.); or microspheres with similar properties to microcarriers, such as glass microspheres, plastic microspheres, etc., can be used as substitutes for microcarriers for testing.

[0288] Example 10

[0289] This example uses the bioreactor of the present invention to carry out iPSC embryoid body perfusion culture experiments.

[0290] The pumping device used in the bioreactor is the 11# pumping device in Example 6, that is, the 11# pumping device uses an elliptical reinforcement component (266 mm, as shown in Figure 20). This embodiment mainly tests the perfusion effect of the elliptical reinforcement component under the iPSC embryoid suspension system culture in a 2L reactor. These geometric dimensions are related to the size of the bioreactor tank. The dimensions designed in this embodiment match the stirred bioreactor (model: FTVS02, supplier: Beijing Huakan Biotechnology Co., Ltd.) used in the embodiment and are not limited as specific parameters.

[0291] Some of the equipment and reagents used in the following examples are as follows:

[0292] mTeSR1 culture medium is a product of STEMCELL Technologies, catalog number: 85850, and is a common commercially available product.

[0293] ACCUTASE is a product of STEMCELL Technologies, product number: 07920, and is a conventional commercially available product.

[0294] PBS solution is a product of Vicente Biotechnology Co., Ltd., product number: 311-010-CL, and is a conventional commercially available product.

[0295] DMEM / F12 culture medium is a product of Gibco, with the product number C11330500BT, and is a conventional commercially available product.

[0296] Y27632 is a product of Sigma, product number: Y0503-5mg, and is a conventional commercially available product.

[0297] iPSC cells are induced pluripotent stem cells, which are common cells in the field.

[0298] The specific experimental operations are as follows.

[0299] 1. Cell seeding: Digest freshly cultured iPSC suspension cells with Accutase, resuspend the cells in mTeSR1 medium containing 10 μM Y27632, and plate at 5*10 5 The cells were inoculated at a density of 1000 × 10 / ml, the culture volume was 800 mL, the reactor parameters were adjusted, and the cells were cultured at 60 rpm in the dark.

[0300] 2. After 24 hours, the perfusion and fluid replacement procedure begins. The outlet of the liquid outlet tube 7 is connected to the peristaltic pump through a silicone hose with an inner diameter of 3.1 mm, and the other end of the hose is connected to a liquid storage container, such as a 3L glass storage bottle.

[0301] 3. Set the peristaltic pump speed to 800 ml / day while the bioreactor is stirred at 60 rpm. Pump liquid continuously during the culture process. At the same time, pump mTeSR1 medium at the same flow rate to complete the perfusion culture.

[0302] Experimental results:

[0303] As shown in Figures 28 and 29, the oval reinforcement component of the liquid extraction device of the present invention can effectively prevent the iPSC embryoid bodies from being extracted when the bioreactor is continuously stirred, and the perfusion culture of iPSC cells can be realized. The iPSC has a rounded and relatively uniform morphology and can proliferate more than 3 times in 4 days. This embodiment shows that the liquid extraction device of the present invention is also applicable to cell clumps such as embryoid bodies.

[0304] The implementation of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and detail, and these are considered to fall within the protection scope of the present invention.

Claims

A liquid pumping device, characterized in that: The invention comprises a liquid extraction tube assembly, wherein the liquid extraction tube assembly is inserted into the tank body of the bioreactor, and the top end of the liquid extraction tube assembly is optionally fixed on the top cover of the bioreactor. The liquid pumping device according to claim 1, characterized in that The liquid extraction tube assembly is detachably connected to the top cover of the bioreactor. The liquid extraction device according to claim 1 or 2, characterized in that: The liquid extraction tube assembly comprises a liquid extraction tube, and the liquid extraction tube comprises a liquid outlet tube body and a main tube body. The liquid outlet tube body is located above the main tube body, and the inner diameter of the liquid outlet tube body is smaller than the inner diameter of the main tube body. The liquid pumping device according to any one of claims 1 to 3, characterized in that: The liquid suction tube assembly further comprises a tapered opening, and the tapered opening is located at the bottom of the main tube body. The liquid extraction device according to any one of claims 3 or 4, characterized in that The liquid extraction tube further comprises a blade fixing rod, which is detachably fixed in the liquid extraction tube and to which a plurality of blades are connected. The liquid pumping device according to claim 5, characterized in that The length of the blade fixing rod is less than or equal to the length of the main pipe. The liquid pumping device according to claim 5 or 6, characterized in that The included angles between the plurality of blades and the blade fixing rod are equal. The liquid pumping device according to any one of claims 5 to 7, characterized in that: The included angle between the plurality of blades and the vertical direction of the blade fixing rod is an acute angle. The liquid pumping device according to any one of claims 5 to 7, characterized in that: The plurality of blades are arranged perpendicularly to the blade fixing rod. The liquid pumping device according to any one of claims 5 to 9, characterized in that: The plurality of blades are evenly distributed on the blade fixing rod. The liquid pumping device according to any one of claims 5 to 10, characterized in that: The multiple blades are divided into multiple blade groups, each blade group includes multiple blades, and the multiple blades of each blade group are located on the same horizontal plane of the main rod through the blade roots and are connected to the blade fixing rod. A bioreactor, characterized in that A liquid extraction device comprising the liquid extraction device according to any one of claims 1 to 11. The bioreactor according to claim 12, characterized in that It also includes a stirring component and a liquid inlet pipe, and the liquid extraction pipe component, the stirring component and the liquid inlet pipe are arranged at intervals. The bioreactor according to claim 13, characterized in that 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. A method for extracting liquid using the liquid extracting device according to any one of claims 1 to 11, characterized in that: The method comprises: inserting a liquid extraction tube assembly into a tank body of a bioreactor, and optionally fixing the top end of the liquid extraction tube assembly on a top cover of the bioreactor; extracting liquid through the liquid extraction device, during which the microtissues entering the liquid extraction tube assembly settle in the liquid extraction tube assembly and eventually detach from the liquid extraction tube assembly to be retained in the tank body, and the culture medium liquid leaves the bioreactor from the top of the liquid extraction tube assembly. The liquid extraction method according to claim 15, characterized in that: The microtissue enters the suction tube through the tapered opening, settles in the main body of the suction tube, and then leaves the suction tube through the tapered opening. The liquid extraction method according to claim 15, characterized in that: The blades prevent the microtissue from moving upward in the main body of the suction tube. The liquid extraction method according to any one of claims 15 to 17, characterized in that: The microtissue is selected from the group consisting of microcarriers, microcarriers containing cells, microcarrier aggregates, microcarrier aggregates containing cells, cell clumps such as embryoid bodies or organoids, and bacterial clumps.

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