Circulation type bioreactor for large-scale cell culture

Abstract

The invention relates to the technical field of biology, in particular to a circulation type bioreactor for large-scale cell culture, which comprises a funnel-shaped container for cell culture, the funnel-shaped container is of a vessel structure with a funnel-shaped bottom and a funnel-shaped top cover; the tip end of the bottom of the funnel-shaped container is of a spherical arched structure or a flat bottom structure; and a supporting device is arranged below the funnel-shaped container and is connected with an eccentric driving device. The eccentric driving device with a certain eccentric amplitude is matched with the funnel-shaped container which is uniquely designed in geometrical shape, so that a mild flow field environment is provided for cell growth, and the process requirements of cell amplification are met.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a circulating bioreactor for large-scale cell culture. Background Art

[0002] With the development of biotechnology, cell and gene therapy has gradually become a treatment method for various types of serious diseases. Among them, the relatively mature one is to use autologous cells to cultivate corresponding therapeutic cells to cure related diseases. However, this therapy cannot be mass-produced, resulting in its high cost that is prohibitive. Thanks to the progress of biotechnology, the bottleneck of allogeneic cell therapy technology has been overcome, making the large-scale and commercial production of therapeutic cells possible. Therefore, the development of devices that can be used for large-scale cell culture in this field will greatly reduce the production cost of drugs and bring a revolutionary change to this industry.

[0003] At present, in the research stage of cell and gene therapy, shake flasks are often used for in vitro cell culture. Although shake flasks can provide cells with a gentle flow field environment and suitable gas exchange, it is difficult to achieve process scale-up, and the culture scale is often only a few dozen milliliters, which cannot be used as a large-scale cell culture method.

[0004] However, the bioreactors commonly used for large-scale cell culture on the market, such as wave reactors, stirred reactors, torrent reactors, airlift reactors, etc., are mainly used in the fields of monoclonal antibodies and vaccines. There is no optimized design for the field of cell and gene therapy, and its flow field is not suitable for large-scale culture of cells in this field.

[0005] Therapeutic cells commonly used in the field of cell and gene therapy, such as stem cells and T cells, are extremely sensitive to shear force compared to cells commonly used in the field of monoclonal antibodies and vaccines. According to research, the shear force generated by bubbles during the bubbling process is sufficient to kill cells and cannot meet the shear force requirements of cell growth. The stirred and airlift reactors commonly used on the market for large-scale cell culture both use bottom bubbling to meet the oxygen requirements of cell growth. Therefore, these two types of reactors cannot be used in the field of cell and gene therapy.

[0006] The reactors that do not require bubbling on the market mainly include wave reactors, torrent reactors, etc. The shear force of this type of reactor is relatively low. The wave reactor uses the up and down swing of the shaker to make the culture medium in the culture bag repeatedly flush the two sides of the bag to mix and transfer mass. However, when the fluid collides with the bag, a large shear force will be generated, causing some cells to die, and ultimately affecting cell growth and expansion. The torrent reactor uses the eccentric rotation of the shaker to mix the fluid in the tank. However, during the mixing process, the fluid collides with the vertical conical surface of the tank, resulting in excessive local shear force and a large range of variation in the turbulent kinetic energy diffusion rate, which affects cell growth and large-scale culture.

[0007] In addition, in the field of cell and gene therapy, in addition to considering cell expansion, the directional differentiation of cells is also one of the important factors that need to be considered. The consistency of cell differentiation will directly affect the quality of the drug. According to research, the dissipation rate of turbulent kinetic energy has a significant effect on the direction of cell differentiation. When the turbulent kinetic energy dissipation rate of the flow field is uniform, the differentiation direction of cells is easy to maintain consistency. Currently, the bioreactors on the market are not specially designed for this feature and are not suitable for large-scale culture of cells in this field.

[0008] The information disclosed in this background technology section is only intended to deepen the understanding of the overall background technology of the present invention, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art already known to those skilled in the art. Summary of the invention

[0009] The object of the present invention is to provide a circulating bioreactor for large-scale cell culture to solve the technical problems existing in the prior art.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a circulating flow bioreactor for large-scale cell culture, which comprises a funnel-shaped container for cell culture; the funnel-shaped container is a vessel structure with a funnel-shaped bottom and a top cover; the bottom tip of the funnel-shaped container is arranged as a spherical arch structure or a flat bottom structure; a supporting device is arranged below the funnel-shaped container, and the supporting device is connected to an eccentric driving device.

[0011] Preferably, the bottom cone angle a of the funnel-shaped container is maintained between 60-150 degrees.

[0012] It is understandable that the shape of the container is not limited thereto, and can be adaptively deformed according to actual needs. For example, the cross-section of the container can be a polygon, or other similar shapes, all of which fall within the protection scope of the present application.

[0013] Preferably, a supporting device is provided below the funnel-shaped container, the upper end of the supporting device is connected to the outer side wall of the funnel-shaped container, and the lower part of the supporting device is connected to the eccentric driving device.

[0014] Preferably, the funnel-shaped container is made of a rigid material, which is suitable for containers of small volume.

[0015] Preferably, the funnel-shaped container is made of a flexible material, and a shape-retaining support body is provided around the funnel-shaped container, which is suitable for large-volume containers.

[0016] The funnel-shaped container may be equipped with a flow-turbulating device, which can be used to increase the mixing effect and adjust the flow field environment, such as shear force, turbulent kinetic energy dissipation rate, etc. The number and form of the flow-turbulating device can be designed according to specific process requirements.

[0017] Preferably, when the container is made of a rigid material, the spoiler can be directly welded to the inner wall of the container.

[0018] Preferably, when the container is made of a flexible material, the spoiler can be mounted on a shape-maintaining support around the container (as shown in the figure).

[0019] Preferably, the eccentric drive device comprises: a shaking table, a six-axis robot, or a combination structure of a drive motor and a drive motor and an eccentric amplitude adjustment device.

[0020] Preferably, the circulating flow bioreactor further comprises: a control system, wherein the control system is communicatively connected to the sensor detection element arranged on the funnel-shaped container.

[0021] Specifically, the container can be connected to a control system, and the values ​​of various process parameters in the container can be collected through sensors configured on the container. Through the control program, in conjunction with pumps, flow meters and other devices, automatic control of physical parameters in the container can be achieved, including but not limited to temperature, rotation speed, PH, DO, gas flow, etc.

[0022] Preferably, the circulating bioreactor further comprises: a filtration system, wherein the filtration system is connected to the funnel-shaped container.

[0023] Specifically, the filtration system includes an alternating flow filtration system, a tangential flow filtration system, a magnetic drive filtration system, etc., to achieve perfusion process culture.

[0024] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention adopts an eccentric driving device with a certain eccentric amplitude, and cooperates with a funnel-shaped container with a uniquely designed geometric shape to provide a gentle flow field environment for cell growth, increase cell culture density, and realize planned culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 A three-dimensional diagram of a circulating bioreactor provided by an embodiment of the present invention; Figure 2 A side view of a loop bioreactor provided by an embodiment of the present invention; Figure 3 A cross-sectional view of a circulating bioreactor provided in an embodiment of the present invention; Figure 4 A schematic diagram of the control principle of a circulating bioreactor provided in an embodiment of the present invention; Figure 5 A schematic diagram of a spoiler device provided in an embodiment of the present invention; Icon: 1- funnel-shaped container; 2- supporting device; 3- driving motor; 4- driven wheel; 5- eccentric amplitude adjustment device. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention. Example

[0029] This embodiment provides a circulating flow bioreactor for large-scale cell culture, which includes a funnel-shaped container for cell culture; the funnel-shaped container is a vessel structure with a funnel-shaped bottom and a top cover; the bottom tip of the funnel-shaped container is set to a spherical arch structure or a flat bottom structure; a supporting device is arranged below the funnel-shaped container, and the supporting device is connected to the eccentric driving device.

[0030] The funnel-shaped container in this embodiment can be understood as a cylindrical container with a certain cone angle, and shapes that are the same or similar to those shown in the figure belong to the protection scope of this application, wherein the cross-section of the container can be circular, polygonal, etc.

[0031] In the present embodiment, preferably, the funnel-shaped container 1 is made of a rigid material, including but not limited to stainless steel or plastic. In addition, the funnel-shaped container 1 can also be made of a flexible material, and a shape-retaining support is provided around the funnel-shaped container 1.

[0032] In this embodiment, the shape of the top cover of the funnel-shaped container 1 can be adjusted according to the actual processing technology, including but not limited to a circular plate shape, a polygonal shape, a dome shape, etc.

[0033] In this embodiment, the bottom and top cover of the funnel-shaped container 1 can include various process interfaces required for the cell culture process, including but not limited to gas exchange devices, air inlet and outlet pipelines, liquid inlet and outlet pipelines, sampling pipelines, pH electrodes, DO electrodes, temperature electrodes, conductivity electrodes, liquid level electrodes, etc.

[0034] In the present embodiment, the diameter and height of the funnel-shaped bottom of the funnel-shaped container 1 are specially designed so that the bottom cone angle a is maintained between 60 and 150 degrees, and the total volume of the container can be enlarged from tens of milliliters to thousands of liters. According to the actual manufacturing process, the tip of the cone bottom can be a spherical arch or a flat bottom. When the tank body of this special design rotates eccentrically, the content fluid can flow around the center along the cone bottom under the action of gravity and centrifugal force. Because there is no collision between the fluid and the tank wall, the overall shear force of the fluid is very gentle. At the same time, in this state of motion, the fluid is spread out at the cone bottom, which can increase the ratio of the area S of the interface between the fluid and the gas to the total volume V of the flow field, which is conducive to the exchange of fluid and gas, and can provide enough oxygen for high-density growth of cells without bubbling.

[0035] In this embodiment, preferably, the eccentric driving device includes: a shaking table, a six-axis robot or a combined structure of a driving motor 3 and an eccentric amplitude adjustment device 5 .

[0036] Combination Figures 1 to 4 As shown, one of the specific forms is used as an example.

[0037] The combined structure of the driving motor 3 and the eccentric shaft specifically includes: a driving motor 3, a driven wheel 4, and an eccentricity amplitude adjusting device 5; three supporting devices 2 are arranged below the funnel-shaped container 1, the upper end of each supporting device 2 is connected to the outer side wall of the funnel-shaped container 1, the middle part of each supporting device 2 is installed in the same horizontal fixed plate, and the lower part of each supporting device 2 is connected to an eccentricity amplitude adjusting device 5. In this way, the three supporting devices 2 are respectively connected to three eccentricity amplitude adjusting devices 5, wherein one eccentricity amplitude adjusting device 5 is connected to the driving motor 3, and the other two eccentricity amplitude adjusting devices 5 are connected to the driven wheel 4. The eccentricity amplitude adjusting device 5 can be manual or electric.

[0038] It can be seen that this embodiment provides a funnel-shaped container within a specific angle range, which, in conjunction with an eccentric drive device with an adjustable eccentric distance, can provide gentle and uniform growth conditions for cell growth. At the same time, this geometric shape can increase the ratio of the solution-gas contact area S to the solution volume V, thereby increasing the oxygen transfer efficiency and achieving high-density cell growth. Example

[0039] The circulating bioreactor provided in this embodiment can be connected to an alternating flow filtration system, a tangential flow filtration system, a magnetic drive filtration system and other filtration systems for use to achieve perfusion process culture. As for other features, they have been described in detail in the above-mentioned embodiment 1. The technical solution disclosed in this embodiment 2 includes the technical solution disclosed in embodiment 1. The content protected by embodiment 1 also belongs to the protection content of this embodiment 2, and the same parts will not be repeated.

[0040] For example, this embodiment provides a filtration system, which uses a mover driven by magnetic force to perform two-phase pumping in a sterile sealed container to create an alternating tangential flow in the filter unit. The filtration system includes: a first filter unit having a first end and a second end and being in fluid communication with the funnel-shaped container; a second filter unit having a first end and a second end and being in fluid communication with the funnel-shaped container; A driver for generating a magnetic field; An alternating flow pump connected to the first ends of the first filter unit and the second filter unit, the alternating flow pump having a mover completely sealed in a pump chamber, the mover being capable of generating a magnetic field coupled with the magnetic field of the driver, the driver driving the mover to reciprocate in the pump chamber by controlling changes in the magnetic field, so as to pump the fluid from the funnel-shaped container through the first filter unit and the second filter unit.

[0041] Preferably, the alternating flow pump generates an alternating tangential flow of the fluid inside the first filter unit and the second filter unit.

[0042] Preferably, the mover has a sealing device to divide the pump chamber of the alternating flow pump into a first chamber and a second chamber; when the mover moves toward the first end direction away from the first filter unit, that is, the α stroke, the alternating flow pump sucks the fluid in the first filter unit and the funnel-shaped container into the first chamber of the alternating flow pump; at the same time, the fluid in the second chamber of the alternating flow pump is discharged into the second filter unit and the funnel-shaped container; when the mover moves toward the first end direction away from the second filter unit, that is, the β stroke, the alternating flow pump sucks the fluid in the second filter unit and the funnel-shaped container into the second chamber of the alternating flow pump; at the same time, the fluid in the first chamber of the alternating flow pump is discharged into the first filter unit and the funnel-shaped container.

[0043] Preferably, the coupling between the magnetic field of the mover of the alternating flow pump and the magnetic field of the driver includes concentric coupling, end surface coupling, or a composite coupling.

[0044] Preferably, the change of the driver magnetic field includes a change in magnetic field flux, a magnetic field displacement, and a magnetic pole conversion.

[0045] Preferably, the sealing device is a liquid sealing device arranged on the mover.

[0046] Preferably, the device further comprises: a controller connected to the driver and configured to control the movement of the driver so as to controllably move the mover.

[0047] Preferably, it further comprises: a first end pressure sensor, the first end pressure sensor being arranged to measure the pressure between the first end of the first filter unit and the second filter unit and the alternating flow pump chamber; and / or, A second end pressure sensor is arranged to measure pressure between the second ends of the first and second filter units and the funnel-shaped container.

[0048] The controller is arranged to control the driver to control the alternating flow pump based on pressure data received from at least one of the first end and second end pressure sensors of the first filter.

[0049] Preferably, it further comprises: a linear encoder connected to the driver for determining the position of the movable driver.

[0050] Preferably, the first filter unit and the second filter unit are each connected to a pipeline branch point via a first permeate pipeline and a second permeate pipeline, and the pipeline branch point is connected to a permeate pump.

[0051] Preferably, the alternating flow pump generates alternating flow in the first permeate pipe and the second permeate pipe, and part of the permeate is returned from the first permeate pipe and the second permeate pipe to the retentate chamber of the filter through the permeate chamber of the filter, thereby backwashing the filter membrane.

[0052] Preferably, when the mover moves in the direction away from the first end of the first filter unit, that is, the α stroke, the alternating flow pump sucks the fluid in the first filter unit and the funnel-shaped container into the first chamber of the alternating flow pump, and the fluid passes through the first permeate pipe and returns to the retentate chamber of the first filter, forming backwashing of the filter membrane; at the same time, the fluid in the second chamber of the alternating flow pump is discharged into the second filter unit and the funnel-shaped container, and permeation is formed; when the mover moves in the direction away from the first end of the second filter unit, that is, the β stroke, the alternating flow pump sucks the fluid in the second filter unit and the funnel-shaped container into the second chamber of the alternating flow pump, and the fluid passes through the second permeate pipe and returns to the retentate chamber of the second filter, forming backwashing of the filter membrane; at the same time, the fluid in the first chamber of the alternating flow pump is discharged into the first filter unit and the funnel-shaped container, and permeation is formed.

[0053] Preferably, the first end of the first filter unit is connected to the funnel-shaped container via a first end check valve, and the second end of the first filter unit is connected to the funnel-shaped container via a second end check valve; the first end of the second filter unit is connected to the funnel-shaped container via the first end check valve; the second end of the second filter unit is fluidly connected to the funnel-shaped container via the second end check valve; the first end check valve is arranged to allow fluid to flow in a direction from the funnel-shaped container to the first end, and to hinder flow in the opposite direction; the second end check valve is arranged to allow fluid to flow in a direction from the second end to the funnel-shaped container and to hinder flow in the opposite direction.

[0054] Preferably, it also includes: the first end check valve and the second end check valve of the first filter unit are respectively connected to a pipeline branch point, and the pipeline branch point is further connected to the funnel-shaped container via a pipeline section; a first end control valve is arranged between the first end of the first filter unit and the pipeline branch point, and a second end control valve is arranged between the second end of the first filter unit and the branch point.

[0055] Preferably, it also includes: the first end check valve and the second end check valve of the second filter unit are respectively connected to a pipeline branch point, and the pipeline branch point is further connected to the funnel-shaped container via a pipeline section; a first end control valve is arranged between the first end of the second filter unit and the pipeline branch point, and a second end control valve is arranged between the second end of the second filter unit and the pipeline branch point.

[0056] Preferably, the alternating flow pump comprises: a main alternating flow pump connected to the first end of the first filter unit and the second filter unit; at least one extended alternating flow pump connected to the first end of the first filter unit and the second filter unit through a manifold; The driver comprises: A main driver, the main driver being adapted to the main alternating flow pump; At least one expansion drive adapted to the expansion alternating flow pump.

[0057] Preferably, the alternating flow pump comprises: a main alternating flow pump connected to the first end of the first filter unit and the second filter unit; at least one gain alternating flow pump connected to the first filter unit and the second end of the second filter unit; The driver comprises: A main driver, the main driver being adapted to the main alternating flow pump; At least one gain driver, the gain driver is adapted to the gain alternating current pump.

[0058] The specific filtering method of this embodiment includes the following steps: S1. adding cell culture medium and cells to the bioreactor, wherein the reactor type includes but is not limited to a stirred reactor, a loop reactor, a wave reactor, etc.; S2. culturing cells in the bioreactor; S3. During the culture period, operating the alternating flow pump, comprising: S31, applying positive pressure to the first end of the first filter unit, discharging the fluid from the pump chamber to the bioreactor chamber via the second end of the first filter unit and the pipe section, during which a portion of the fluid is guided to enter the permeate chamber of the first filter unit via the semi-permeable wall portion of the first filter unit; and simultaneously, applying negative pressure to the first end of the second filter unit, sucking the fluid from the bioreactor chamber to the pump chamber via the pipe section and the second end of the second filter unit, during which a portion of the fluid is guided to enter the permeate chamber via the semi-permeable wall portion of the second filter unit; S32, and reverse the direction of the force applied by the alternating flow pump, so that the second end of the first filter unit draws fluid from the bioreactor chamber through the pipe segment, and at the same time, the second end of the second filter unit discharges fluid to the bioreactor chamber through the pipe segment.

[0059] S33, repeating steps S31 and S32 at least once, wherein the permeate chamber, the reactor chamber and the alternating flow pump chamber are part of the same closed filtration system; S4. Aspirating permeate from the permeate outlet port.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A circulating bioreactor for large-scale cell culture, characterized in that: The invention comprises a funnel-shaped container for cell culture; the funnel-shaped container is a vessel structure with a funnel-shaped bottom and a top cover; the bottom tip of the funnel-shaped container is arranged as a spherical arch structure or a flat bottom structure; a supporting device is arranged below the funnel-shaped container, and the supporting device is connected to an eccentric driving device.

2. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: The bottom cone angle a of the funnel-shaped container is maintained between 60 and 150 degrees.

3. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: A supporting device is arranged below the funnel-shaped container, the supporting device is connected to the outer side wall of the funnel-shaped container, and the lower part of the supporting device is connected to the eccentric driving device.

4. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: The funnel-shaped container is made of a rigid material.

5. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: The part of the funnel-shaped container in contact with the culture medium is made of a flexible material, and a shape-maintaining support body is provided around the funnel-shaped container.

6. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: The funnel-shaped container is equipped with a flow disturbance device.

7. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: The funnel-shaped container is provided with process interfaces that meet the process requirements during cell culture, including but not limited to liquid inlet and outlet, gas inlet and outlet, sampling, standby, and sensor interfaces.

8. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: The eccentric driving device comprises: a mechanical shaking table or a magnetic suspension shaking table, a six-axis robot or a combined structure of a driving motor and an eccentricity amplitude regulating device.

9. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: Also includes: A control system is communicatively connected with a sensor detection element arranged on the funnel-shaped container.

10. The circulating bioreactor for large-scale cell culture according to claim 1, characterized in that: Also includes: A filtration system is connected to the funnel-shaped container.

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